Anti-kit antibodies and uses thereof

ABSTRACT

Provided herein, in one aspect, are antibodies that immunospecifically bind to a human KIT antigen comprising the fourth and/or fifth extracellular Ig-like domains (that is, D4 and/or D5 domains), polynucleotides comprising nucleotide sequences encoding such antibodies, and expression vectors and host cells for producing such antibodies. The antibodies can inhibit KIT activity, such as ligand-induced receptor phosphorylation. Also provided herein are kits and pharmaceutical compositions comprising antibodies that specifically bind to a KIT antigen, as well as methods of treating or managing a KIT-mediated disorder or disease and methods of diagnosing a KIT-mediated disorder or disease using the antibodies described herein.

This application claims the benefit of U.S. Provisional Application No.61/436,483 filed on Jan. 26, 2011; U.S. Provisional Application No.61/507,430 filed on Jul. 13, 2011; and U.S. Provisional Application No.61/537,482 filed on Sep. 21, 2011, each of which is hereby incorporatedby reference in its entirety.

1. FIELD

Provided herein are antibodies that specifically bind to a KIT antigen,polynucleotides comprising nucleotide sequences encoding suchantibodies, expression vectors and host cells for producing suchantibodies, kits and pharmaceutical compositions comprising antibodiesthat immunospecifically bind to a KIT antigen, methods for treating ormanaging a KIT-mediated disorder, and diagnostic methods.

2. BACKGROUND

KIT (or c-Kit) is a type III receptor tyrosine kinase encoded by thec-kit gene. KIT comprises five extracellular immunoglobulin (Ig)-likedomains, a single transmembrane region, an inhibitory cytoplasmicjuxtamembrane domain, and a split cytoplasmic kinase domain separated bya kinase insert segment (see, e.g., Yarden et al., Nature, 1986,323:226-232; Ullrich and Schlessinger, Cell, 1990, 61:203-212; Cliffordet al., J. Biol. Chem., 2003, 278:31461-31464). The human c-kit geneencoding the KIT receptor has been cloned as described by Yarden et al.,EMBO J., 1987, 6:3341-3351. KIT is also known as CD117 or stem cellfactor receptor (“SCFR”), because it is the receptor for the stem cellfactor (“SCF”) ligand (also known as Steel Factor or Kit Ligand). SCFligand binding to the first three extracellular Ig-like domains of KITinduces receptor dimerization, and thereby activates intrinsic tyrosinekinase activity through the phosphorylation of specific tyrosineresidues in the juxtamembrane and kinase domains (see, e.g., Weiss andSchlessinger, Cell, 1998, 94:277-280; Clifford et al., J. Biol. Chem.,2003, 278:31461-31464). Members of the Stat, Src, ERK, and AKT signalingpathways have been shown to be downstream signal transducers of KITsignaling.

The fourth (D4) and fifth (D5) extracellular Ig-like domains of KIT arebelieved to mediate receptor dimerization (see, e.g., InternationalPatent Application Publication No. WO 2008/153926; Yuzawa et al., Cell,2007, 130:323-334).

Expression of KIT has been detected in various cell types, such as mastcells, stem cells, brain cells, melanoblasts, ovary cells, and cancercells (e.g., leukemia cells). Studies of loss-of-function KIT mutationsindicate that KIT is important for the normal growth of hematopoieticprogenitor cells, mast cells, melanocytes, primordial germ cells, andthe interstitial cells of Cajal (see, e.g., Besmer, P., Curr. Opin. CellBiol., 1991, 3:939-946; Lyman et al., Blood, 1998, 91:1101-1134; Ashman,L. K., Int. J. Biochem. Cell Biol., 1999, 31:1037-1051; Kitamura et al.,Mutat. Res., 2001, 477:165-171; Mol et al., J. Biol. Chem., 2003,278:31461-31464). Moreover, KIT plays an important role inhematopoiesis, melanogenesis, and gametogenesis (see Ueda et al., Blood,2002, 99:3342-3349).

Abnormal KIT activity has been implicated in connection with a number ofcancers. For example, gain-of-function KIT mutations resulting inSCF-independent, constitutive activation of KIT are found in certaincancer cells and are associated with certain cancers such as leukemia(e.g., chronic myelogenous leukemia) and gastrointestinal stromal tumors(see, e.g., Mol et al., J. Biol. Chem., 2003, 278:31461-31464).

3. SUMMARY

Provided herein, in one aspect, are antibodies (and antigen-bindingfragments thereof) that immunospecifically bind to a D4/D5 region in theextracellular domain of KIT (e.g., human KIT) and inhibit a KITactivity, as well as related compositions, reagents and methods.

In a specific embodiment, provided herein is antibody 37M (includingantigen-binding fragments thereof) comprising a variable light (“VL”)chain region comprising the amino acid sequence of SEQ ID NO: 2, and avariable heavy (“VH”) chain region comprising the amino acid sequence ofSEQ ID NO: 3. In a particular embodiment, provided herein is antibody37C (including antigen-binding fragments thereof) comprising a VL chainregion comprising the amino acid sequence of SEQ ID NO: 2 and a VH chainregion comprising the amino acid sequence of SEQ ID NO: 5. In otherparticular embodiments, presented herein is an antibody (or anantigen-binding fragment thereof) comprising the CDRs of antibody 37M or37C (e.g., VL CDRs of SEQ ID NO: 2 and/or VH CDRs of SEQ ID NO: 3 or 5;or SEQ ID NOs: 20, 21, 22, 23, 24, and/or 25). In certain embodiments,provided herein are antibodies (or antigen-binding fragments thereof)which compete with antibody 37M or 37C for binding to a D4/D5 region ofKIT (e.g., human KIT), for example SEQ ID NO: 15. In certainembodiments, provided herein is an antibody (or an antigen-bindingfragment thereof) that immunospecifically binds to a D4/D5 region of KIT(e.g., human KIT), for example SEQ ID NO: 15, wherein the antibody bindsto the same epitope as an epitope of antibody 37M or 37C.

In one aspect, provided herein is an antibody (or an antigen-bindingfragment thereof), which immunospecifically binds to a D4/D5 region ofhuman KIT (e.g., SEQ ID NO: 15), comprising:

-   -   (A) a variable light (“VL”) chain region comprising the amino        acid sequence of SEQ ID NO: 2, and a variable heavy (“VH”) chain        region comprising the amino acid sequence of SEQ ID NO: 3; or    -   (B) a VL chain region comprising the amino acid sequence of SEQ        ID NO: 2 and a VH chain region comprising the amino acid        sequence of SEQ ID NO: 5.        In a particular embodiment, an antibody (or an antigen-binding        fragment thereof), which immunospecifically binds to a D4/D5        region of human KIT (e.g., SEQ ID NO: 15), comprises: a VL chain        region comprising the amino acid sequence of SEQ ID NO: 2, and a        VH chain region comprising the amino acid sequence of SEQ ID NO:        3 or SEQ ID NO: 3 with one amino acid substitution at the        C-terminal amino acid (i.e., amino acid at position 116 of SEQ        ID NO: 3). In a specific embodiment, the amino acid substitution        at position 116 of SEQ ID NO: 3 is an S to A substitution.

In a second aspect, provided herein is an antibody (or anantigen-binding fragment thereof), which immunospecifically binds to aD4/D5 region of human KIT (e.g., SEQ ID NO: 15), comprising:

-   -   (i) a VL chain region comprising a VL CDR1, VL CDR2, and VL CDR3        having the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21,        and SEQ ID NO: 22, respectively; and (ii) a VH chain region        comprising a VH CDR1, VH CDR2, and VH CDR3 having the amino acid        sequences of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25,        respectively.

In a third aspect, provided herein is an antibody (or an antigen-bindingfragment thereof), which immunospecifically binds to a D4/D5 region ofhuman KIT (e.g., SEQ ID NO: 15), comprising:

-   -   a VL chain region comprising a VL CDR1, VL CDR2, and VL CDR3        having the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21,        and SEQ ID NO: 22, respectively; and    -   a VH chain region comprising the amino acid sequence of SEQ ID        NO: 3 or 5.

In a fourth aspect, provided herein is an antibody (or anantigen-binding fragment thereof), which immunospecifically binds to aD4/D5 region of human KIT (e.g., SEQ ID NO: 15), comprising:

-   -   a VL chain region comprising the amino acid sequence of SEQ ID        NO: 2; and a VH chain region comprising a VH CDR1, VH CDR2, and        VH CDR3 having the amino acid sequences of SEQ ID NO:23, SEQ ID        NO: 24, and SEQ ID NO: 25, respectively.

In certain embodiments, antibodies described herein comprise a humanlight chain constant region and a human heavy chain constant region. Infurther embodiments, the human light chain constant region is a humankappa light chain constant region. In another embodiment, the humanheavy chain constant region is a human gamma heavy chain constantregion. In certain embodiments, the antibody is an IgG1 isotype fantibody. In particular embodiments, the human light chain constantregion comprises the amino acid sequence of SEQ ID NO: 12. In specificembodiments, the human heavy chain constant region comprises the aminoacid sequence of SEQ ID NO: 13. In certain embodiments, an antibodydescribed herein comprises (i) a human light chain constant regioncomprising the amino acid sequence of SEQ ID NO: 12, (ii) a variablelight chain region comprising the amino acid sequence of SEQ ID NO: 2,(iii) a human heavy chain constant region comprising the amino acidsequence of SEQ ID NO: 13, and (iv) a variable heavy chain regioncomprising the amino acid sequence of SEQ ID NO: 3 or 5. In certainembodiments, an antibody described herein (or an antigen-bindingfragment thereof) comprises a light chain comprising the amino acidsequence of SEQ ID NO: 6 (or the amino acid sequence of SEQ ID NO: 6starting at position 20, lacking the signal peptide), and a heavy chaincomprising the amino acid sequence of SEQ ID NO: 7 (or the amino acidsequence of SEQ ID NO: 7 starting at position 20, lacking the signalpeptide).

In a particular embodiment, the antibody described herein (or anantigen-binding fragment thereof) is a monoclonal antibody, e.g., amurine, chimeric, or humanized monoclonal antibody. In a certainembodiment, the antibody described herein (or an antigen-bindingfragment thereof) is an isolated antibody. In a particular embodiment,the antibody described herein (or an antigen-binding fragment thereof)is a chimeric antibody. In another embodiment, the antibody describedherein (or an antigen-binding fragment thereof) is a humanized antibodycomprising the CDRs of antibody 37M or 37C (e.g., VL CDRs of SEQ ID NO:2 and/or VH CDRs of SEQ ID NO: 3 or 5; or SEQ ID NOs: 20, 21, 22, 23,24, and/or 25). In another particular embodiment, the antibody describedherein is an antigen-binding antibody fragment, e.g., a Fab antibody(e.g., a Fab antibody of antibody 37M or 37C). In yet another particularembodiment, the antibody described herein (or the antigen-bindingfragment thereof) is a human IgG1 or IgG4 antibody. In yet anotherparticular embodiment, the antibody described herein (or anantigen-binding fragment thereof) is an inhibitor of KIT activity. In afurther embodiment, the antibody described herein (or an antigen-bindingfragment thereof) which is an inhibitor of KIT activity, inhibits KITreceptor phosphorylation by at least 25% as determined by a solid phaseELISA assay. In one embodiment, the antibody described herein (or anantigen-binding fragment thereof) which is an inhibitor of KIT activityinhibits KIT receptor phosphorylation by 25% to 80% as determined by asolid phase ELISA assay. In another embodiment, the antibody describedherein (or an antigen-binding fragment thereof) which is an inhibitor ofKIT activity inhibits KIT receptor phosphorylation by at least 50% asdetermined by a solid phase ELISA assay. In yet another embodiment, anantibody described herein (or an antigen-binding fragment thereof) whichis an inhibitor of KIT activity does not block KIT ligand binding to theKIT receptor. In a further embodiment, the antibody described herein (oran antigen-binding fragment thereof) which is an inhibitor of KITactivity does not inhibit KIT receptor dimerization. In yet a furtherembodiment, the antibody described herein (or an antigen-bindingfragment thereof) which is an inhibitor of KIT activity enhances KITreceptor internalization or KIT receptor degradation. In anotherembodiment, an antibody described herein (or an antigen-binding fragmentthereof) which is an inhibitor of KIT activity induces apoptosis when acell expressing KIT is contacted with an effective amount of theantibody.

In a specific embodiment, an antibody provided herein (or anantigen-binding fragment thereof), which immunospecifically binds to aD4/D5 region of human KIT (e.g., SEQ ID NO: 15), immunospecificallybinds to the same epitope as that of an antibody comprising a VL domaincomprising the amino acid sequence of SEQ ID NO: 2, and a VH domaincomprising the amino acid sequence of SEQ ID NO: 3 or 5. In a particularembodiment, an antibody provided herein (or an antigen-binding fragmentthereof), which immunospecifically binds to a D4/D5 region of human KIT(e.g., SEQ ID NO: 15), immunospecifically binds to the same epitope asthat of an antibody comprising (i) a VL chain region comprising VL CDR1,VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 20,21, and 22, respectively, and (ii) a VH chain region comprising VH CDR1,VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 23,24, and 25, respectively. In a certain embodiment, an antibody providedherein (or an antigen-binding fragment thereof), whichimmunospecifically binds to a D4/D5 region of human KIT (e.g., SEQ IDNO: 15), immunospecifically binds to the same epitope as that ofantibody 37M or 37C.

In a specific embodiment, an antibody provided herein (or anantigen-binding fragment thereof), which immunospecifically binds to aD4/D5 region of human KIT (e.g., SEQ ID NO: 15), competes (e.g., in adose-dependent manner) for binding to a D4/D5 region of human KIT withan antibody comprising a VL domain comprising the amino acid sequence ofSEQ ID NO: 2, and a VH domain comprising the amino acid sequence of SEQID NO: 3 or 5. In a particular embodiment, an antibody provided herein(or an antigen-binding fragment thereof), which immunospecifically bindsto a D4/D5 region of human KIT (e.g., SEQ ID NO: 15 competes (e.g., in adose-dependent manner) for binding to a D4/D5 region of human KIT withan antibody comprising (i) a VL chain region comprising VL CDR1, VLCDR2, and VL CDR3 having the amino acid sequences of SEQ ID NOs: 20, 21,and 22, respectively, and (ii) a VH chain region comprising VH CDR1, VHCDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 23, 24,and 25, respectively. In a certain embodiment, an antibody providedherein (or an antigen-binding fragment thereof), whichimmunospecifically binds to a D4/D5 region of human KIT (e.g., SEQ IDNO: 15), competes (e.g., in a dose-dependent manner) for binding to aD4/D5 region of human KIT with antibody 37M or 37C. In a specificembodiment, an antibody provided herein (or an antigen-binding fragmentthereof) which immunospecifically binds to a D4/D5 region of human KIT(e.g., SEQ ID NO: 15), and competes (e.g., in a dose-dependent manner)for binding to a D4/D5 region of human KIT with antibody 37M or 37C,comprises (i) VL CDR1, VL CDR2, and VL CDR3 having the amino acidsequences of SEQ ID NOs: 20, 21, and 22, respectively, and (ii) a VHchain region comprising VH CDR1, VH CDR2, and VH CDR3 having the aminoacid sequences of SEQ ID NOs: 23, 24, and 25, respectively.

In certain embodiments, an antibody described herein or antigen-bindingfragment thereof (e.g., antibody comprising VL CDR1, VL CDR2, and VLCDR3 having the amino acid sequence of SEQ ID NO: 20, 21, and 22,respectively, and/or a VH chain region comprising VH CDR1, VH CDR2, andVH CDR3 having the amino acid sequence of SEQ ID NO: 23, 24, and 25,respectively) binds to an extracellular domain of human KIT comprising amutation, for example a somatic mutation associated with cancer (e.g.,GIST), such as a mutation in exon 9 of human KIT wherein the Ala and Tyrresidues at positions 502 and 503 are duplicated. In certainembodiments, an antibody described herein or antigen-binding fragmentthereof (e.g., antibody comprising VL CDR1, VL CDR2, and VL CDR3 havingthe amino acid sequence of SEQ ID NO: 20, 21, and 22, respectively,and/or a VH chain region comprising VH CDR1, VH CDR2, and VH CDR3 havingthe amino acid sequence of SEQ ID NO: 23, 24, and 25, respectively)binds to an extracellular domain of wild-type human KIT and anextracellular domain of human KIT comprising a mutation, for example asomatic mutation associated with cancer (e.g., GIST), such as a mutationin exon 9 of human KIT wherein the Ala and Tyr residues at positions 502and 503 are duplicated. In certain embodiments, an antibody describedherein or antigen-binding fragment thereof (e.g., antibody comprising VLCDR1, VL CDR2, and VL CDR3 having the amino acid sequence of SEQ ID NO:20, 21, and 22, respectively, and/or a VH chain region comprising VHCDR1, VH CDR2, and VH CDR3 having the amino acid sequence of SEQ ID NO:23, 24, and 25, respectively) binds to an extracellular domain of humanKIT which is glycosylated. In certain embodiments, an antibody describedherein or antigen-binding fragment thereof (e.g., antibody comprising VLCDR1, VL CDR2, and VL CDR3 having the amino acid sequence of SEQ ID NO:20, 21, and 22, respectively, and/or a VH chain region comprising VHCDR1, VH CDR2, and VH CDR3 having the amino acid sequence of SEQ ID NO:23, 24, and 25, respectively) binds to an extracellular domain of humanKIT which is not glycosylated.

In a eighth aspect, provided herein is a vector, e.g., a mammalianexpression vector comprising one or more polynucleotides (or isolatedpolynucleotides) comprising nucleotide sequences encoding an antibodydescribed herein (e.g., antibody 37M or 37C, or an antigen-bindingfragment thereof, or an antibody comprising CDRs of antibody 37M or37C). In a particular embodiment, provided herein is an expressionvector (e.g., mammalian expression vector) comprising a polynucleotide(or isolated polynucleotide) comprising nucleotide sequences encoding aVL chain region and/or a VH chain region of an antibody described herein(e.g., antibody 37M or 37C, or an antigen-binding fragment thereof, oran antibody comprising CDRs of antibody 37M or 37C). In a particularembodiment, a polynucleotide comprises a nucleotide sequence of SEQ IDNO: 8 encoding a VL chain region. In a particular embodiment, apolynucleotide comprises the nucleotide sequence of SEQ ID NO: 9encoding a VH chain region. In a particular embodiment, a polynucleotidecomprises the nucleotide sequence of SEQ ID NO: 10 encoding a lightchain. In a particular embodiment, a polynucleotide comprises thenucleotide sequence of SEQ ID NO: 11 encoding a heavy chain.

In a particular embodiment, a polynucleotide comprises a nucleotidesequence that has 85% sequence identity to SEQ ID NO: 8 and encodes a VLchain region of an antibody that immunospecifically binds to the D4/D5region of KIT. In a particular embodiment, a polynucleotide comprises anucleotide sequence that has 85% sequence identity to SEQ ID NO: 9 andencodes a VH chain region of an antibody that immunospecifically bindsto the D4/D5 region of KIT. In a particular embodiment, a polynucleotidecomprises a nucleotide sequence that has 85% sequence identity to SEQ IDNO: 10 and encodes a light chain of an antibody that immunospecificallybinds to the D4/D5 region of KIT. In a particular embodiment, apolynucleotide comprises a nucleotide sequence that has 85% sequenceidentity to SEQ ID NO: 11 and encodes a heavy chain of an antibody thatimmunospecifically binds to the D4/D5 region of KIT.

In a ninth aspect, provided herein is a host cell comprising a vectordescribed herein, e.g., a mammalian expression vector. In certainembodiments, a cell described herein comprises one or morepolynucleotides comprising nucleotide sequences encoding an antibodyprovided herein which immunospecifically binds to a D4/D5 region ofhuman KIT (e.g., antibody 37M or 37C, or an antigen-binding fragmentthereof, or an antibody comprising the CDRs of antibody 37M or 37C). Ina particular embodiment, a cell described herein comprises one or morepolynucleotides comprising nucleotide sequences encoding a VL chainregion and a VH chain region of an antibody provided herein whichimmunospecifically binds to a D4/D5 region of human KIT (e.g., antibody37M or 37C, or an antigen-binding fragment thereof, or an antibodycomprising the CDRs of antibody 37M or 37C).

In a tenth aspect, provided herein is a hybridoma cell producing anantibody described herein (e.g., antibody 37M, or an antigen-bindingfragment thereof, or an antibody comprising CDRs of antibody 37M).

In a particular aspect, provided herein is a method of making anantibody which immunospecifically binds to a D4/D5 region of human KIT(SEQ ID NO: 15) comprising culturing the host cell or hybridoma celldescribed herein. In a specific embodiment, such method of making anantibody further comprises the step of purifying the antibody from saidhost cell or hybridoma cell.

In a specific aspect, provided herein is a method of making an antibodywhich immunospecifically binds to a D4/D5 region of human KIT (SEQ IDNO: 15) comprising administering to an animal (e.g., a non-human animal,such as a mouse or rat) a human KIT antigen having the amino acidsequence of: (i) SEQ ID NO: 14; (ii) SEQ ID NO: 14 without the first 25amino acids (signal peptide); (iii) SEQ ID NO: 14 without the first25-33 amino acids; or (iv) SEQ ID NO: 15, optionally with a 5×His tag atthe C-terminus and/or 1-8 amino acids at the N-terminus, wherein theanimal produces an antibody which immunospecifically binds to a D4/D5region of human KIT (SEQ ID NO: 15). In a specific embodiment, suchmethod further comprises the step of obtaining cells which produce anantibody which immunospecifically binds to a D4/D5 region of human KIT(SEQ ID NO: 15) from the animal. In a certain embodiment, such methodfurther comprises the step of generating hybridoma cells from the cellsobtained from the animal. In a particular embodiment, such methodfurther comprises the step of obtaining one or more polynucleotidesencoding the antibody or fragment thereof (e.g., VH domain and/or VLdomain) from the animal or cells of the animal. In a particularembodiment, such method comprises the step of cloning one or morepolynucleotides encoding the antibody or fragment thereof (e.g., VHdomain and/or VL domain) from the animal or cells of the animal into avector (e.g., an expression vector).

In a particular aspect, provided herein are isolated or purified KITpolypeptides having the amino acid sequence of: (i) SEQ ID NO: 14; (ii)SEQ ID NO: 14 without the first 25 amino acids (signal peptide); (iii)SEQ ID NO: 14 without the first 25-33 amino acids; or (iv) SEQ ID NO:15, optionally with a 5×His tag at the C-terminus and/or 1-8 amino acidsat the N-terminus, wherein the KIT polypeptides comprise less than theentire extracellular domain of KIT (e.g., human KIT). In a certainembodiment, provided herein is a composition comprising such isolated orpurified KIT polypeptides.

In an eleventh aspect, provided herein is a pharmaceutical compositioncomprising an antibody described herein (e.g., antibody 37M or 37C, oran antigen-binding fragment thereof, or an antibody comprising CDRs ofantibody 37M or 37C) and a pharmaceutically acceptable carrier.

In a twelfth aspect, provided herein is a kit comprising an antibodydescribed herein (e.g., antibody 37M or 37C, or an antigen-bindingfragment thereof, or an antibody comprising CDRs of antibody 37M or37C).

In a thirteenth aspect, provided herein is a method for treating ormanaging a KIT-mediated disorder or disease in a subject in needthereof, comprising administering to the subject a therapeuticallyeffective amount of an antibody described herein (e.g., antibody 37M or37C, or an antigen-binding fragment thereof, or an antibody comprisingCDRs of antibody 37M or 37C). In a particular embodiment, theKIT-mediated disorder or disease is cancer, an inflammatory condition,or fibrosis. In another particular embodiment, the cancer is leukemia,chronic myelogenous leukemia, lung cancer, small cell lung cancer,melanoma, sarcoma, or gastrointestinal stromal tumors. In yet anotherparticular embodiment, the cancer is refractory to treatment by atyrosine kinase inhibitor. In a further particular embodiment, thetyrosine kinase inhibitor is GLEEVEC™ (imatinib mesylate) or SUTENT®(sunitinib).

In a particular embodiment, provided herein is a method for treating ormanaging a KIT-mediated disorder or disease in a subject in needthereof, comprising administering to the subject (i) an antibodydescribed herein (e.g., antibody 37M or 37C, or an antigen-bindingfragment thereof, or an antibody comprising CDRs of antibody 37M or37C), and (ii) a second therapeutic agent. In a specific embodiment, thesecond therapeutic agent is a small molecule kinase inhibitor (e.g.,imatinib mesylate or sunitinib). In a specific embodiment, the secondtherapeutic agent is a histone deacetylase inhibitor (e.g., vorinostator suberoylanilide hydroxamic acid (SAHA)). In a particular embodiment,an antibody (or antigen-binding fragment) which immunospecifically bindsto a D4/D5 region of human KIT for use in the methods provided herein isconjugated to an agent (e.g., a chemotherapeutic agent, a toxic agent, adetectable agent).

In an fourteenth aspect, provided herein is a method for diagnosing asubject with a KIT-mediated disorder or disease comprising contacting asample obtained from the subject with an antibody described herein(e.g., antibody 37M or 37C, or an antigen-binding fragment thereof, oran antibody comprising CDRs of antibody 37M or 37C) and detecting theexpression level of KIT in the sample. In a particular embodiment, theantibody is conjugated to a detectable molecule. In another particularembodiment, the detectable molecule is an enzyme, fluorescent molecule,luminescent molecule, or radioactive molecule.

In a fifteenth aspect, provided herein is a method for inhibiting KITactivity in a cell expressing KIT comprising contacting the cell with aneffective amount of an antibody described herein (e.g., antibody 37M or37C, or an antigen-binding fragment thereof, or an antibody comprisingCDRs of antibody 37M or 37C).

In a sixteenth aspect, provided herein is a method for inducing orenhancing apoptosis in a cell expressing KIT comprising contacting thecell with an effective amount of an antibody described herein (e.g.,antibody 37M or 37C, or an antigen-binding fragment thereof, or anantibody comprising CDRs of antibody 37M or 37C).

In a seventeenth aspect, provided herein is a method for inducing celldifferentiation comprising contacting a cell expressing KIT with aneffective amount of an antibody described herein (e.g., anantigen-binding fragment thereof, antibody 37M or 37C, or an antibodycomprising CDRs of antibody 37M or 37C). In one embodiment, the cell isa stem cell.

In a particular aspect, provided herein is a conjugate comprising anagent linked (e.g., directly or via a linker) to an antibody describedherein (or an antigen-binding fragment thereof), which antibodyimmunospecifically binds to a D4/D5 region of human KIT (e.g., SEQ IDNO: 15) and comprises:

-   -   (i) a VL chain region comprising a VL CDR1, VL CDR2, and VL CDR3        having the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21,        and SEQ ID NO: 22, respectively; and (ii) a VH chain region        comprising a VH CDR1, VH CDR2, and VH CDR3 having the amino acid        sequences of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25,        respectively.

In certain embodiments, the agent conjugated to an anti-KIT antibody isa toxin (e.g., abrin, ricin A, pseudomonas exotoxin, cholera toxin, ordiphtheria toxin). In particular embodiments, the conjugate isinternalized by a cell (e.g., cell expressing KIT protein). In specificembodiments, the conjugate comprises an agent linked via a linker to ananti-KIT antibodies. In certain embodiments, the conjugate comprises anagent linked directly to an anti-KIT antibodies. In particularembodiments, the conjugate comprises an agent linked covalently to ananti-KIT antibody. In certain embodiments, the conjugate comprises anagent linked non-covalently to an anti-KIT antibody. In specificembodiments, the methods provided herein (e.g., method for treatingcancer) comprise administering a conjugate described herein to anindividual in need thereof.

3.1 Terminology

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art.

As used herein and unless otherwise specified, the terms “about” or“approximately” mean within plus or minus 10% of a given value or range.

As used herein and unless otherwise specified, “administer” or“administration” refers to the act of injecting or otherwise physicallydelivering a substance (e.g., an anti-KIT antibody provided herein) to asubject or a patient, such as by mucosal, topical, intradermal,intravenous, intramuscular delivery and/or any other method of physicaldelivery described herein or known in the art.

As used herein and unless otherwise specified, the terms “antibody” and“immunoglobulin” and “Ig” are terms of art and can be usedinterchangeably herein and refer to a molecule with an antigen bindingsite that immunospecifically binds an antigen.

As used herein and unless otherwise specified, an “antigen” is a moietyor molecule that contains an epitope, and, as such, also is specificallybound by antibody. In a specific embodiment, the antigen, to which anantibody described herein binds, is KIT (e.g., human KIT), or a fragmentthereof, for example, an extracellular domain of KIT (e.g., human KIT)or a D4/D5 region of KIT (e.g., human KIT).

As used herein and unless otherwise specified, an “epitope” is a term inthe art and refers to a localized region of an antigen to which anantibody can specifically bind. A region or a polypeptide contributingto an epitope can be contiguous amino acids of the polypeptide or anepitope can come together from two or more non-contiguous regions of thepolypeptide.

As used herein and unless otherwise specified, the terms “antigenbinding domain,” “antigen binding region,” “antigen binding fragment,”and similar terms refer to a portion of an antibody molecule whichcomprises the amino acid residues that interact with an antigen andconfer on the antibody molecule its specificity for the antigen (e.g.,the complementarity determining regions (CDR)). The antigen bindingregion can be derived from any animal species, such as rodents (e.g.,mouse, rat or hamster) and humans. The CDRs of an antibody molecule canbe determined by any method well known to one of skill in the art. Inparticular, the CDRs can be determined according to the Kabat numberingsystem (see Kabat et al. (1991) Sequences of proteins of immunologicalinterest. (U.S. Department of Health and Human Services, Washington,D.C.) 5^(th) ed.)_(.)

As used herein and unless otherwise specified, a “conservative aminoacid substitution” is one in which the amino acid residue is replacedwith an amino acid residue having a side chain with a similar charge.Families of amino acid residues having side chains with similar chargeshave been defined in the art. These families include amino acids withbasic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine).

As used herein and unless otherwise specified, a “conformationalepitope” or “nonlinear epitope” or “discontinuous epitope” refers to onecomprised of at least two amino acids which are not consecutive aminoacids in a single protein chain. For example, a conformational epitopecan be comprised of two or more amino acids which are separated by astretch of intervening amino acids but which are close enough to berecognized by an antibody (e.g., an anti-KIT antibody) described hereinas a single epitope. As a further example, amino acids which areseparated by intervening amino acids on a single protein chain, or aminoacids which exist on separate protein chains, can be brought intoproximity due to the conformational shape of a protein structure orcomplex to become a conformational epitope which can be bound by ananti-KIT antibody described herein. It will be appreciated by one ofskill in the art that, in general, a linear epitope bound by an anti-KITantibody described herein may or may not be dependent on the secondary,tertiary, or quaternary structure of the KIT receptor. For example, insome embodiments, an anti-KIT antibody described herein binds to a groupof amino acids regardless of whether they are folded in a natural threedimensional protein structure. In other embodiments, an anti-KITantibody described herein does not recognize the individual amino acidresidues making up the epitope, and require a particular conformation(bend, twist, turn or fold) in order to recognize and bind the epitope.

As used herein and unless otherwise specified, the term “constantregion” or “constant domain” refers to an antibody portion, e.g., acarboxyl terminal portion of a light and/or heavy chain which is notdirectly involved in binding of an antibody to antigen but whichexhibits various effector functions, such as interaction with the Fcreceptor. The terms refer to a portion of an immunoglobulin moleculehaving a generally more conserved amino acid sequence relative to animmunoglobulin variable domain.

As used herein and unless otherwise specified, the terms “D4/D5 region”or “D4/D5 domain” refer to a region within a KIT polypeptide spanningthe fourth Ig-like extracellular (“D4”) domain, the fifth Ig-likeextracellular (“D5”) domain, and the hinge region in between the D4 andD5 domains (“D4-D5 hinge region”), of KIT, in the following order fromthe amino terminus to the carboxyl terminus: D4, D4-D5 hinge region, andD5. As used herein, amino acids V308 to H515 of FIG. 1 and thepolypeptide depicted at FIG. 2A herein are considered a D4/D5 region ordomain.

As used herein and unless otherwise specified, the terms “KIT” or “KITreceptor” or “KIT polypeptide” refer to any form of full-length KITincluding, but not limited to, native KIT, an isoform of KIT, aninterspecies KIT homolog, or a KIT variant, e.g., naturally occurring(for example, allelic or splice variant, or mutant, e.g., somaticmutant) or artificially constructed variant (for example, a recombinantor chemically modified variant). KIT is a type III receptor tyrosinekinase encoded by the c-kit gene (see, e.g., Yarden et al., Nature,1986, 323:226-232; Ullrich and Schlessinger, Cell, 1990, 61:203-212;Clifford et al., J. Biol. Chem., 2003, 278:31461-31464; Yarden et al.,EMBO J., 1987, 6:3341-3351; Mol et al., J. Biol. Chem., 2003,278:31461-31464). GenBank™ accession number NM 000222 provides anexemplary human KIT nucleic acid sequence. GenBank™ accession numbers NP001087241, P10721, and AAC50969 provide exemplary human KIT amino acidsequences. GenBank™ accession number AAH75716 provides an exemplarymurine KIT amino acid sequence. Native KIT comprises five extracellularimmunoglobulin (Ig)-like domains (D1, D2, D3, D4, D5), a singletransmembrane region, an inhibitory cytoplasmic juxtamembrane domain,and a split cytoplasmic kinase domain separated by a kinase insertsegment (see, e.g., Yarden et al., Nature, 1986, 323:226-232; Ullrichand Schlessinger, Cell, 1990, 61:203-212; Clifford et al., J. Biol.Chem., 2003, 278:31461-31464). An exemplary amino acid sequence of theD4/D5 region of human KIT is provided in FIG. 1, at amino acid residuesV308 to H515. In a specific embodiment, KIT is human KIT. In aparticular embodiment, KIT can exist as a monomer, dimer, multimer,native form, or denatured form.

As used herein and unless otherwise specified, the terms “effectiveamount” or “therapeutically effective amount” refer to an amount of atherapy (e.g., an antibody or pharmaceutical composition providedherein) which is sufficient to reduce and/or ameliorate the severityand/or duration of a given disease and/or a symptom related thereto.These terms also encompass an amount necessary for the reduction oramelioration of the advancement or progression of a given disease,reduction or amelioration of the recurrence, development or onset of agiven disease, and/or to improve or enhance the prophylactic ortherapeutic effect(s) of another therapy (e.g., a therapy other than ananti-KIT antibody provided herein). In some embodiments, “effectiveamount” as used herein also refers to the amount of an antibodydescribed herein to achieve a specified result (e.g., inhibition (e.g.,partial inhibition) of a KIT biological activity of a cell, such asinhibition of cell proliferation or cell survival, or enhancement orinduction of apoptosis or cell differentiation).

In the context of a peptide or a polypeptide, the term “fragment” asused herein refers to a peptide or polypeptide that comprises less thanfull length amino acid sequence. Such a fragment can arise, for example,from a truncation at the amino terminus, a truncation at the carboxyterminus, and/or an internal deletion of a residue(s) from the aminoacid sequence. Fragments can, for example, result from alternative RNAsplicing or from in vivo protease activity. In certain embodiments, KITfragments or antibody fragments (e.g., antibody fragments thatimmunospecifically bind to a KIT polypeptide) include polypeptidescomprising an amino acid sequence of at least 5 contiguous amino acidresidues, at least 10 contiguous amino acid residues, at least 15contiguous amino acid residues, at least 20 contiguous amino acidresidues, at least 25 contiguous amino acid residues, at least 40contiguous amino acid residues, at least 50 contiguous amino acidresidues, at least 60 contiguous amino residues, at least 70 contiguousamino acid residues, at least 80 contiguous amino acid residues, atleast 90 contiguous amino acid residues, at least contiguous 100 aminoacid residues, at least 125 contiguous amino acid residues, at least 150contiguous amino acid residues, at least 175 contiguous amino acidresidues, at least 200 contiguous amino acid residues, or at least 250contiguous amino acid residues of the amino acid sequence of a KITpolypeptide or an antibody (e.g., antibody that immunospecifically bindto a KIT polypeptide), respectively. In a specific embodiment, afragment of a KIT polypeptide or an antibody (e.g., antibody thatimmunospecifically bind to a KIT polypeptide) retains at least 1, atleast 2, or at least 3 functions of the polypeptide or antibody.

As used herein and unless otherwise specified, the term “heavy chain”when used in reference to an antibody refers to any distinct types,e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (μ), based onthe amino acid sequence of the constant domain, which give rise to IgA,IgD, IgE, IgG and IgM classes of antibodies, respectively, includingsubclasses of IgG, e.g., IgG₁, IgG₂, IgG₃ and IgG₄. In a specificembodiment, the heavy chain is a human heavy chain.

As used herein and unless otherwise specified, the term “host cell”refers to a particular cell that comprises an exogenous nucleic acidmolecule, e.g., a cell that has been transfected or transformed with anucleic acid molecule, and the progeny or potential progeny of such aparent cell. Progeny of such a cell may not be identical to the parentcell due to mutations or environmental influences that can occur insucceeding generations or integration of the nucleic acid molecule intothe host cell genome.

As used herein and unless otherwise specified, the terms“immunospecifically binds,” “immunospecifically recognizes,”“specifically binds,” and “specifically recognizes” are analogous termsin the context of antibodies and refer to molecules that specificallybind to an antigen (e.g., epitope or immune complex) as understood byone skilled in the art. For example, a molecule that specifically bindsto an antigen can bind to other peptides or polypeptides, generally withlower affinity as determined by, e.g., immunoassays, Biacore™, KinExA3000 instrument (Sapidyne Instruments, Boise, Id.), or other assaysknown in the art. In a specific embodiment, molecules thatimmunospecifically bind to an antigen bind to the antigen with a K_(a)that is at least 2 logs, 2.5 logs, 3 logs, 4 logs or greater than theK_(a) when the molecules bind to another antigen. In another specificembodiment, molecules that immunospecifically bind to an antigen do notcross react with other proteins. In another specific embodiment,molecules that immunospecifically bind to an antigen do not cross reactwith other non-KIT proteins.

As used herein and unless otherwise specified, the term “in combination”in the context of the administration of other therapies refers to theuse of more than one therapy. The use of the term “in combination” doesnot restrict the order in which therapies are administered. Thetherapies may be administered, e.g., serially, sequentially,concurrently, or concomitantly.

As used herein and unless otherwise specified, an “isolated” or“purified” antibody is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which theantibody is derived, or substantially free of chemical precursors orother chemicals when chemically synthesized.

As used herein and unless otherwise specified, an “isolated”polynucleotide or nucleic acid molecule is one which is separated fromother nucleic acid molecules which are present in the natural source(e.g., in a human) of the nucleic acid molecule. Moreover, an “isolated”nucleic acid molecule, such as a cDNA molecule, can be substantiallyfree of other cellular material, or culture medium when produced byrecombinant techniques, or substantially free of chemical precursors orother chemicals when chemically synthesized. For example, the language“substantially free” includes preparations of polynucleotide or nucleicacid molecule having less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1%(in particular less than about 10%) of other material, e.g., cellularmaterial, culture medium, other nucleic acid molecules, chemicalprecursors and/or other chemicals. In a specific embodiment, a nucleicacid molecule(s) encoding an antibody described herein is isolated orpurified.

The terms “Kabat numbering,” and like terms are recognized in the artand refer to a system of numbering amino acid residues in the heavy andlight chain variable regions of an antibody, or an antigen bindingportion thereof (Kabat et al. (1971) Ann. NY Acad. Sci. 190:382-391 and,Kabat et al. (1991) Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242). Using the Kabat numbering system, CDRs withinan antibody heavy chain molecule are typically present at amino acidpositions 31 to 35 (“CDR1”), amino acid positions 50 to 65 (“CDR2”), andamino acid positions 95 to 102 (“CDR3”). Using the Kabat numberingsystem, CDRs within an antibody light chain molecule are typicallypresent at amino acid positions 24 to 34 (CDR1), amino acid positions 50to 56 (CDR2), and amino acid positions 89 to 97 (CDR3).

As used herein and unless otherwise specified, the term “light chain”when used in reference to an antibody refers to any distinct types,e.g., kappa (κ) of lambda (λ) based on the amino acid sequence of theconstant domains. Light chain amino acid sequences are well known in theart. In specific embodiments, the light chain is a human light chain.

As used herein and unless otherwise specified, the terms “manage,”“managing,” and “management” refer to the beneficial effects that asubject derives from a therapy (e.g., a prophylactic or therapeuticagent), which does not result in a cure of a KIT-mediated disease ordisorder. In certain embodiments, a subject is administered one or moretherapies (e.g., prophylactic or therapeutic agents, such as an antibodydescribed herein) to “manage” a KIT-mediated disease (e.g., cancer,inflammatory condition, or fibrosis), one or more symptoms thereof, soas to prevent the progression or worsening of the disease.

As used herein and unless otherwise specified, the term “monoclonalantibody” refers to an antibody obtained from a population of homogenousor substantially homogeneous antibodies, and each monoclonal antibodywill typically recognize a single epitope on the antigen. In specificembodiments, a “monoclonal antibody,” as used herein, is an antibodyproduced by a single hybridoma or other cell (e.g., host cell producinga recombinant antibody), wherein the antibody immunospecifically bindsto a KIT epitope (e.g., an epitope of a D4/D5 region of human KIT) asdetermined, e.g., by ELISA or other antigen-binding or competitivebinding assay known in the art or in the Examples provided herein.Generally, a population of monoclonal antibodies can be generated bycells, a population of cells, or a cell line. The term “monoclonal” isnot limited to any particular method for making the antibody. Forexample, monoclonal antibodies described herein can be made by thehybridoma method as described in Kohler et al.; Nature, 256:495 (1975)or can be isolated from phage libraries using the techniques asdescribed herein, for example. Other methods for the preparation ofclonal cell lines and of monoclonal antibodies expressed thereby arewell known in the art (see, for example, Chapter 11 in: Short Protocolsin Molecular Biology, (2002) 5th Ed., Ausubel et al., eds., John Wileyand Sons, New York).

As used herein and unless otherwise specified, the term“pharmaceutically acceptable” means being approved by a regulatoryagency of the Federal or a state government, or listed in the U.S.Pharmacopeia, European Pharmacopeia or other generally recognizedPharmacopeia for use in animals, and more particularly in humans.

As used herein and unless otherwise specified, the term “polyclonalantibodies” refers to an antibody population that includes a variety ofdifferent antibodies directed to the same and to different epitopeswithin an antigen or antigens. Methods for producing polyclonalantibodies are known in the art (See, e.g., see, for example, Chapter 11in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel etal., eds., John Wiley and Sons, New York).

As used herein and unless otherwise specified, the terms “impede” or“impeding” in the context of a KIT-mediated disorder or disease refer tothe total or partial inhibition (e.g., less than 100%, 95%, 90%, 80%,70%, 60%, 50%, 40%, 30%, 20%, 10%, or 5%) or blockage of thedevelopment, recurrence, onset or spread of a KIT-mediated diseaseand/or symptom related thereto, resulting from the administration of atherapy or combination of therapies provided herein (e.g., a combinationof prophylactic or therapeutic agents, such as an antibody describedherein).

As used herein and unless otherwise specified, the term “prophylacticagent” refers to any agent that can totally or partially inhibit thedevelopment, recurrence, onset or spread of a KIT-mediated diseaseand/or symptom related thereto in a subject. In certain embodiments, theterm “prophylactic agent” refers to an antibody described herein. Incertain other embodiments, the term “prophylactic agent” refers to anagent other than an antibody described herein. Generally, a prophylacticagent is an agent which is known to be useful to or has been or iscurrently being used to prevent a KIT-mediated disease and/or a symptomrelated thereto or impede the onset, development, progression and/orseverity of a KIT-mediated disease and/or a symptom related thereto. Inspecific embodiments, the prophylactic agent is a human anti-KITantibody, such as a humanized or a fully human anti-KIT monoclonalantibody.

As used herein and unless otherwise specified, the term “recombinanthuman antibody” includes human antibodies that are prepared, expressed,created or isolated by recombinant means, such as antibodies expressedusing a recombinant expression vector transfected into a host cell,antibodies isolated from a recombinant, combinatorial human antibodylibrary, antibodies isolated from an animal (e.g., a mouse, rabbit,goat, or cow) that is transgenic and/or transchromosomal for humanimmunoglobulin genes (see e.g., Taylor, L. D. et al. (1992) Nucl. AcidsRes. 20:6287-6295) or antibodies prepared, expressed, created orisolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies can have variable and constant regions derived fromhuman germline immunoglobulin sequences. In certain embodiments, theamino acid sequences of such recombinant human antibodies have beenmodified such thus the amino acid sequences of the VH and/or VL regionsof the recombinant antibodies are sequences that, while derived from andrelated to human germline VH and VL sequences, do not naturally existwithin the human antibody germline repertoire in vivo.

As used herein and unless otherwise specified, the term “side effects”encompasses unwanted and adverse effects of a therapy (e.g., aprophylactic or therapeutic agent). Unwanted effects are not necessarilyadverse. An adverse effect from a therapy (e.g., a prophylactic ortherapeutic agent) can be harmful or uncomfortable or risky. Examples ofside effects include, diarrhea, cough, gastroenteritis, wheezing,nausea, vomiting, anorexia, abdominal cramping, fever, pain, loss ofbody weight, dehydration, alopecia, dyspenea, insomnia, dizziness,mucositis, nerve and muscle effects, fatigue, dry mouth, and loss ofappetite, rashes or swellings at the site of administration, flu-likesymptoms such as fever, chills and fatigue, digestive tract problems andallergic reactions. Additional undesired effects experienced by patientsare numerous and known in the art. Many are described in the Physician'sDesk Reference (63^(rd) ed., 2009).

As used herein and unless otherwise specified, the terms “subject” and“patient” are used interchangeably. As used herein, a subject ispreferably a mammal such as a non-primate (e.g., cows, pigs, horses,cats, dogs, goats, rabbits, rats, mice, etc.) or a primate (e.g., monkeyand human), most preferably a human. In one embodiment, the subject is amammal, preferably a human, having a KIT-mediated disorder or disease.In another embodiment, the subject is a mammal, preferably a human, atrisk of developing a KIT-mediated disorder or disease. In anotherembodiment, the subject is a non-human primate.

As used herein and unless otherwise specified, the terms “therapies” and“therapy” can refer to any protocol(s), method(s), compositions,formulations, and/or agent(s) that can be used in the prevention,treatment, management, or amelioration of a condition or disorder orsymptom thereof (e.g., cancer or one or more symptoms or conditionassociated therewith; inflammatory condition or one or more symptoms orcondition associated therewith; fibrosis or one or more symptoms orcondition associated therewith). In certain embodiments, the terms“therapies” and “therapy” refer to drug therapy, adjuvant therapy,radiation, surgery, biological therapy, supportive therapy, and/or othertherapies useful in treatment, management, prevention, or ameliorationof a condition or disorder or one or more symptoms thereof (e.g., canceror one or more symptoms or condition associated therewith; inflammatorycondition or one or more symptoms or condition associated therewith;fibrosis or one or more symptoms or condition associated therewith). Incertain embodiments, the term “therapy” refers to a therapy other thanan anti-KIT antibody described herein or pharmaceutical compositionthereof. In specific embodiments, an “additional therapy” and“additional therapies” refer to a therapy other than a treatment usingan anti-KIT antibody described herein or pharmaceutical composition. Ina specific embodiment, a therapy includes the use of an anti-KITantibody described herein as an adjuvant therapy. For example, using ananti-KIT antibody described herein in conjunction with a drug therapy,biological therapy, surgery, and/or supportive therapy.

As used herein and unless otherwise specified, the term “therapeuticagent” refers to any agent that can be used in the treatment, managementor amelioration of a KIT-mediated disease and/or a symptom relatedthereto. In certain embodiments, the term “therapeutic agent” refers toan antibody described herein. In certain other embodiments, the term“therapeutic agent” refers to an agent other than an antibody describedherein. Preferably, a therapeutic agent is an agent which is known to beuseful for, or has been or is currently being used for the treatment,management or amelioration of a KIT-mediated disease or one or moresymptoms related thereto. In specific embodiments, the therapeutic agentis a human anti-KIT antibody, such as a fully human anti-KIT monoclonalantibody.

As used herein and unless otherwise specified, the terms “treat,”“treatment” and “treating” refer to the reduction or amelioration of theprogression, severity, and/or duration of a KIT-mediated disease (e.g.,cancer, inflammatory disorder, or fibrosis) resulting from theadministration of one or more therapies (including, but not limited to,the administration of one or more prophylactic or therapeutic agents,such as an antibody provided herein).

As used herein and unless otherwise specified, the terms “variableregion” or “variable domain” refer to a portion of an antibody,generally, a portion of a light or heavy chain, typically about theamino-terminal 110 to 120 amino acids in the mature heavy chain andabout 90 to 100 amino acids in the mature light chain, which differextensively in sequence among antibodies and are used in the binding andspecificity of a particular antibody for its particular antigen. Thevariability in sequence is concentrated in those regions calledcomplementarity determining regions (CDRs) while the more highlyconserved regions in the variable domain are called framework regions(FR). Without wishing to be bound by any particular mechanism or theory,it is believed that the CDRs of the light and heavy chains are primarilyresponsible for the interaction of the antibody with antigen. In aspecific embodiment, numbering of amino acid positions of antibodiesdescribed herein is according to the EU Index, as in Kabat et al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242(“Kabat et al.”). In certain embodiments, the variable region is a humanvariable region. In certain embodiments, the variable region comprisesrodent or murine CDRs and human framework regions (FRs). In particularembodiments, the variable region is a primate (e.g., non-human primate)variable region. In certain embodiments, the variable region comprisesrodent or murine CDRs and primate (e.g., non-human primate) frameworkregions (FRs).

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the amino acid sequence of full length human KIT (SEQ IDNO: 1), GenBank™ accession number AAC50969. The first through fifthextracellular Ig-like domains (i.e., D1, D2, D3, D4, and D5) areindicated; “{” depicts the amino-terminal residue of each domain and “}”depicts the carboxyl-terminal residue of each domain. The D1 domain isdepicted at P34 to R112, the D2 domain is depicted at D113 to P206, theD3 domain is depicted at A207 to D309, the D4 domain is depicted at K310to N410, the hinge region between D4 and D5 is located at V409 to N410,and the D5 domain is depicted at T411 to K509. Also, the D1/D2 hingeregion is located at D113 to L117; the D2/D3 hinge region is located atP206 to A210; and the D3/D4 hinge region is located at D309 to G311. TheD4/D5 region comprises K310 to K509. The transmembrane domain comprisesresidues F525 to Q545, and the kinase domain comprises residues K589 toS933.

FIG. 2A depicts the amino acid sequence of the recombinant KIT D4/D5polypeptide used to generate antibodies. Human KIT amino acids V308 toH515 (SEQ ID NO: 15) are depicted in bold. The recombinant KIT D4/D5polypeptide depicted (SEQ ID NO: 14) contains (i) the first 33 aminoacids (i.e., M1 to E33) of the amino terminus of human KIT (includingthe signal peptide (underlined, not bold) which is cleaved duringprocessing and, as such, was not utilized as part of the antigen used togenerate antibodies), (ii) the D4/D5 region of human KIT (bold), and(iii) a 5×His tag (italics) at the carboxyl terminus.

FIG. 2B depicts the amino acid sequence of the recombinant D4 domainpolypeptide used in binding assays (see, e.g., section 6). Inparticular, the recombinant D4 domain polypeptide comprising residuesV308 to K412 (SEQ ID NO: 17), depicted as bold, of human KIT includesthe D4 domain (residues K310 to N410). The recombinant D4 domainpolypeptide amino acid sequence depicted (SEQ ID NO: 16) contains (i)the first 33 amino acids of the amino terminus of human KIT (includingthe signal peptide (underlined, not bold) which is cleaved duringprocessing and, as such, was not utilized as part of the polypeptideused in binding assays), (ii) the D4 domain (bold, single-underlined) ofhuman KIT, and (iii) a 6×His tag (italics) at the carboxyl terminus.

FIG. 2C depicts the amino acid sequence of the recombinant D5 domainpolypeptide used in binding assays (see, e.g., section 6). Inparticular, the recombinant D5 domain polypeptide comprising residuesN410 to H515 (SEQ ID NO: 19), depicted as bold, of human KIT includesthe D5 region (residues T411 to K509) of human KIT. The recombinant D5domain polypeptide amino acid sequence depicted (SEQ ID NO: 18) contains(i) the first 33 amino acids of the amino terminus of human KIT(including the signal peptide (underlined, not bold) which is cleavedduring processing and, as such, was not utilized as part of thepolypeptide used in binding assays), (ii) the D5 domain (bold,double-underlined) of human KIT, and (iii) a 5×His tag (italics) at thecarboxyl terminus.

FIG. 3A depicts the variable light chain region amino acid sequence ofanti-KIT antibodies 37M and 37C (SEQ ID NO: 2). The framework regions(FR1, FR2, FR3, and FR4), and CDRs (CDR1, CDR2, and CDR3) are indicated.

FIG. 3B depicts the variable heavy chain region amino acid sequence ofanti-KIT antibodies 37M (SEQ ID NO: 3), and 37C (SEQ ID NO: 5). Theframework regions (FR1, FR2, FR3, and FR4), and CDRs (CDR1, CDR2, andCDR3) are indicated.

FIG. 4 depicts the amino acid sequences of the VL domain (SEQ ID NO: 2)and VH domain (SEQ ID NO: 3) of anti-KIT antibody 37M as well as the DNAsequences encoding the VL domain (SEQ ID NO: 8) and the VH domain (SEQID NO:9). The CDRs (CDR1, CDR2, and CDR3) are bold and underlined.

FIG. 5A depicts the amino acid sequences of the light chain (SEQ ID NO:6) and the heavy chain (SEQ ID NO: 7) of antibody 37C. The VL domain(SEQ ID NO: 2) comprises amino acid residues D20-E124 of the lightchain, and the VH domain (SEQ ID NO: 5) comprises amino acid residuesQ20-A135 of the heavy chain. The CDRs (CDR1, CDR2, and CDR3) areunderlined. The amino acid sequences of the light chain constant domain(SEQ ID NO: 12) and of the heavy chain constant domain (SEQ ID NO: 13)are depicted in bold. The light chain constant domain (SEQ ID NO: 12)corresponds to amino acid residues T128 to C233 of SEQ ID NO: 6. Theheavy chain constant domain (SEQ ID NO: 13) corresponds to amino acidresidues A136 to K465 of SEQ ID NO: 7. The signal peptides are doubleunderlined. The signal peptide of the light chain comprises the aminoacid sequence MGWSCIILFL VATATGVHS (SEQ ID NO: 43). The signal peptideof the heavy chain comprises the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 44). These signal peptides are removed duringpost-translational processing to yield the mature forms of the lightchain and heavy chain.

FIG. 5B depicts the DNA sequence encoding the light chain of antibody37C (SEQ ID NO: 10), and its corresponding amino acid sequence (SEQ IDNO: 6).

FIG. 5C depicts the DNA sequence encoding the heavy chain of antibody37C (SEQ ID NO: 11), and its corresponding amino acid sequence (SEQ IDNO: 7).

FIG. 6A depicts the binding activity of antibody 37M to the D4/D5 regionof human KIT, as well as to either the D4 domain or D5 domain of humanKIT by solid phase ELISA.

FIG. 6B depicts the binding activity of antibody 37C to the D4/D5 regionof human KIT by solid phase ELISA.

FIG. 7 depicts the activity of antibody 37M on CHO cells exogenouslyexpressing wild-type human KIT (CHO/KIT-WT; right panel). Parental CHOcells not exogenously expressing wild-type human KIT (CHO parental; leftpanel) were used as controls.

FIG. 8 depicts the inhibition of phosphorylation of the cytoplasmicdomain of human KIT by antibodies 37M and 37C by cell-basedphosphorylation assays. The 50% inhibition concentration ofphosphorylation for 37M and 37C in these assays are calculated to be 109pM and 167 pM, respectively.

FIG. 9 depicts a bar graph showing that antibody 37M can inhibit colonyformation by CHO/KIT-WT cells in soft agar assays. The control sample(“ctrl”) included CHO/KIT-WT cells not exposed to any antibody.

FIG. 10A shows the sort scheme for CHO/KIT-WT cells. P5 indicates thegated population of sorted cells that stained positive for KITexpression with a fluorescent signal intensity of ≥10⁴. Gating based onforward scatter (FSC) and side scatter (SSC) is also depicted.

FIG. 10B depicts a graph showing flow cytometry data for CHO parentalcells, which are untransfected CHO cells, incubated with variousconcentrations of antibody 37M, an anti-KIT antibody (CD117), or ananti-IgG antibody (negative control) prior to processing for flowcytometry analysis.

FIG. 10C depicts a graph showing flow cytometry data for CHO cellsrecombinantly expressing wild-type KIT (CHO/KIT-WT) which were incubatedwith various concentrations of antibody 37M, an anti-KIT antibody(CD117), or an anti-IgG antibody (negative control) prior to processingfor flow cytometry analysis.

FIG. 11A depicts results from cell-based phosphorylation assays usingCHO/KIT-WT cells demonstrating the ability of antibody 37M to inhibitKIT phosphorylation. The graph plots relative luminescence units (RLUs)against log concentration (M) of antibody 37M. The 50% inhibitionconcentration of phosphorylation for 37M in these assays was calculatedto be approximately 96 pM.

FIG. 11B depicts results from cell-based phosphorylation assays usingsorted (high KIT expression) CHO/KIT-WT cells demonstrating the abilityof antibody 37M or 37C to inhibit KIT phosphorylation. The depictedgraph plots relative luminescence units (RLUs) versus log concentration(M) of either antibody 37M or antibody 37C. The IC₅₀ values of antibody37M and antibody 37C were calculated to be approximately 315 pM and 334pM, respectively.

FIG. 12 depicts a graph showing flow cytometry data for CHO cellsexpressing a mutated form of KIT wherein the Ala and Tyr residues atpositions 502 and 503, respectively, are duplicated (CHO 502.503). TheCHO cells expressing the KIT duplication mutation were incubated withvarious concentrations of antibody 37M, an anti-KIT antibody (CD117), oran isotype control antibody (negative control) prior to processing forflow cytometry analysis.

FIG. 13 depicts results from cell-based phosphorylation assays usingsorted (high KIT expression) CHO/KIT-WT cells demonstrating the abilityof antibody 37M to inhibit AKT phosphorylation. The depicted graph plotsrelative luminescence units (RLUs) versus log concentration (M) ofantibody 37M. The IC₅₀ value of antibody 37M was calculated to beapproximately 138 pM.

FIG. 14A depicts the results from competition binding assays (solidphase ELISA) where antibody 37M served as the reference antibody andantibody 37C served as the competitor antibody (10 nM), and the antigenis the KIT D4/D5 antigen depicted in FIG. 2A. The symbol “●” representsdata points for antibody 37M binding affinity to the KIT D4/D5 antigen,at various concentrations and without competitor antibody 37C, and thesymbol “▪” represents data points for antibody 37M binding affinity tothe KIT D4/D5 antigen, at various concentrations and with competitorantibody 37C (10 nM). The EC₅₀ value for reference antibody 37M, whichwas calculated to be 115 pM, increased to 2.8 nM in the presence of thecompetitor antibody 37C (10 nM).

FIG. 14B depicts the results from competition binding assays whereantibody 37C served as the reference antibody and antibody 37M served asthe competitor antibody (10 nM), and the antigen is the KIT D4/D5antigen depicted in FIG. 2A. The symbol “●” represents data points forantibody 37C binding affinity to the KIT D4/D5 antigen, at variousconcentrations and without competitor antibody 37M, and the symbol “▪”represents data points for antibody 37C binding affinity to the KITD4/D5 antigen, at various concentrations and with competitor antibody37M (10 nM). As shown in FIG. 14B, the EC₅₀ value for reference antibody37C, which was calculated to be 48 pM, increased to 1 nM in the presenceof competitor antibody 37M (10 nM).

FIGS. 15A-D depict data from experiments evaluating the effects of anantibody drug conjugate (ADC) targeting KIT protein on cellproliferation. In FIGS. 15A (GIST430 cells) and 15C (GIST 48B cells),the symbols “▴,” “▪,” and “●” represent data points for cells exposed toantibody 37M and 0 ng, 25 ng, and 50 ng of saporin-conjugated secondaryantibody (“saporin”), respectively. In FIGS. 15B (GIST430 cells) and D(GIST 48B cells), the symbols “▴,” “▪,” and “●” represent data pointsfor cells exposed to an anti-VEGFR-2 monoclonal antibody (anti-VEGFR-2mAb) and 0 ng, 25 ng, and 50 ng of saporin-conjugated secondary antibody(“saporin”), respectively.

FIGS. 16A-F depict data from experiments evaluating the effects of anantibody drug conjugate (ADC) targeting KIT protein on proliferation ofcells expressing wild-type KIT or mutant KIT. FIGS. 16A-B depict resultsfrom experiments with CHO cells engineered to express wild-type KIT(CHO/WT-KIT). FIGS. 16C-D depict results from experiments with CHO cellsengineered to express mutant KIT containing a duplication of the Ala andTyr residues at positions 502 and 503 (CHO/KIT-502.503)/FIGS. 16E-Fdepict results from experiments with CHO cells engineered to expressmutant KIT containing V560D and Y823D amino acid substitutions. In FIGS.16A, C and E, the symbols “▴,” “▪,” and “●” represent data points forcells exposed to antibody 37M and 0 ng, 25 ng, and 50 ng ofsaporin-conjugated secondary antibody (“saporin”), respectively. InFIGS. 16B, D, and F, the symbols “▴,” “▪,” and “●” represent data pointsfor cells exposed to antibody 37M and 0 ng, 25 ng, and 50 ng ofsaporin-conjugated secondary antibody (“saporin”), respectively.

FIG. 17A depicts a gel showing KIT antigen, containing the entireextracellular domain (KIT ECD) or domains D4 and D5 (KIT D4/D5), in itsglycosylated form (“Control”) and de-glycosylated form due to exposureto the endoglycosidase N-Glycosidase F (PNGase F).

FIG. 17B depicts the binding activity of antibody 37M to glycosylatedKIT D4/D5 antigen (“●”; “D4-D5-His”) and de-glycosylated KIT D4/D5antigen (“▪”; “D4-D5-His PNGase F”) (see FIGS. 2A and 17A) by solidphase ELISA.

FIG. 17C depicts the binding activity of antibody 37M to glycosylated(“●”; “ECD-His”) and de-glycosylated (“▪”; “ECD-His PNGase F”) KITantigen containing the entire extracellular domain (see FIG. 17A) bysolid phase ELISA.

FIG. 17D depicts a Western blot of KIT protein immunoprecipitated byantibody 37M from lysate of CHO cells expressing wild-type KIT(CHO/WT-KIT) Immunoprecipitated KIT protein was untreated or treatedwith PNGase F prior to electrophoresis. A Western blot for beta-Tubulinprotein was performed as a control.

5. DETAILED DESCRIPTION

Provided herein are antibodies (e.g., murine, chimeric or humanizedantibodies), including antigen-binding fragments thereof, thatimmunospecifically bind to a KIT polypeptide (e.g., a KIT polypeptidecontaining the D4/D5 region of human KIT). Also provided are isolatednucleic acids (polynucleotides) encoding such antibodies (e.g., murine,chimeric, or humanized antibodies), and antigen-binding fragmentsthereof. Further provided are vectors and cells (e.g., host cells)comprising nucleic acids encoding such antibodies or antigen-bindingfragments thereof. Also provided are methods of making such antibodies,cells, e.g., hybridoma cells, and antibodies produced by such cells,e.g., hybridoma cells. Also provided herein is a method of treating ormanaging a KIT-mediated disorder or disease (e.g., cancer, inflammatorycondition, or fibrosis) or one or more effects of such KIT-mediateddisorder or disease comprising administering one or more antibodiesdescribed herein, or an antigen-binding fragment thereof. Also providedherein is a method of diagnosing a KIT-mediated disorder or disease(e.g., cancer, inflammatory condition, or fibrosis) comprisingcontacting a sample with one or more antibodies (or antigen-bindingfragment thereof) described herein and determining the expression levelof KIT in the sample relative to a reference sample (e.g., a controlsample). Further provided herein is a method for inhibiting KIT activityin a cell expressing KIT comprising contacting the cell with aneffective amount of an antibody or antibodies described herein or anantigen-binding fragment thereof. Also further provided herein is amethod for inducing or enhancing cell differentiation or apoptosis in acell expressing KIT comprising contacting the cell with an effectiveamount of an antibody or antibodies described herein.

5.1 Antibodies

In specific aspects, provided herein are isolated antibodies (includingantigen-binding fragments thereof) that immunospecifically bind to theD4/D5 region of KIT, e.g., human KIT. Amino acid residues V308 to H515(SEQ ID NO: 15) of FIGS. 1 and 2 represent an exemplary D4/D5 region ofhuman KIT. In a specific embodiment, an antibody described herein (or anantigen-binding fragment thereof) immunospecifically binds to a D4domain of KIT, e.g., human KIT, for example, amino acid residues K310 toN410 of human KIT (see FIGS. 1, 2A and 2B). In another specificembodiment, an antibody described herein (or an antigen-binding fragmentthereof) immunospecifically binds to a D5 domain of KIT, e.g., humanKIT, for example, amino acid residues V409 to N410 of human KIT (seeFIGS. 1, 2A and 2C) with lower affinity than to a D4 domain of KIT,e.g., human KIT. In a particular embodiment, an antibody describedherein (or an antigen-binding fragment thereof) immunospecifically bindsto a D4 domain of KIT, e.g., human KIT, with higher affinity than to aD5 domain of KIT, e.g., human KIT, for example, the higher affinity isat least 10 fold, 20 fold, 50 fold, 100 fold, 500 fold, or 1000 fold asdetermined by methods known in the art, e.g., ELISA or Biacore assays.

In a specific embodiment, an antibody described herein (or anantigen-binding fragment thereof) immunospecifically binds to a D4/D5region of KIT, e.g., human KIT, and has higher affinity for a KITantigen containing the D4/D5 region than for a KIT antigen consistingessentially of a D4 domain only or a KIT antigen consisting essentiallyof a D5 domain only. In a particular embodiment, an antibody describedherein (or an antigen-binding fragment thereof) immunospecifically bindsto a D4/D5 region of KIT, e.g., human KIT, and has at least 2 fold, 3fold, 4 fold, 5 fold, or 10 fold higher affinity for a KIT antigencontaining the D4/D5 region than for a KIT antigen consistingessentially of a D4 domain only. In a particular embodiment, an antibodydescribed herein (or an antigen-binding fragment thereof)immunospecifically binds to a D4/D5 region of KIT, e.g., human KIT, andhas approximately a 2 fold to 3 fold higher affinity for a KIT antigencontaining the D4/D5 region than for a KIT antigen consistingessentially of a D4 domain only. In a particular embodiment, an antibodydescribed herein (or an antigen-binding fragment thereof)immunospecifically binds to a D4/D5 region of KIT, e.g., human KIT, andhas at least 5 fold, 10 fold, 50 fold, 100 fold, or 1,000 fold higheraffinity for a KIT antigen containing the D4/D5 region than for a KITantigen consisting essentially of a D5 domain only.

In a particular embodiment, an antibody described herein (or anantigen-binding fragment thereof) immunospecifically binds to a KITantigen comprising or consisting essentially of the amino acid sequenceof SEQ ID NO: 14 or 15. In a specific embodiment, an antibody describedherein (or an antigen-binding fragment thereof) immunospecifically bindsto a D4 domain of KIT, e.g., human KIT. In a particular embodiment, anantibody described herein immunospecifically binds to a KIT antigencomprising or consisting essentially of the amino acid sequence of SEQID NO: 16 or 17. In another specific embodiment, an antibody describedherein (or an antigen-binding fragment thereof) immunospecifically bindsa D4/D5 region of KIT, e.g., human KIT, wherein the antibody has higheraffinity to a D4 domain of KIT than to a D5 domain of KIT, e.g., humanKIT, as determined by methods known in the art, e.g., ELISA or Biacoreassay. In another specific embodiment, an antibody described herein (oran antigen-binding fragment thereof) immunospecifically binds a D4/D5region of KIT, e.g., human KIT, wherein the antibody has higher affinityto a D4 domain of KIT than to a D5 domain of KIT, e.g., human KIT, asdetermined by methods known in the art, e.g., ELISA or Biacore assay,and wherein the higher affinity is at least 2 fold, 5 fold, 10 fold, 50fold, 100 fold, or 500 fold. In another specific embodiment, an antibodydescribed herein (or an antigen-binding fragment thereof)immunospecifically binds a D4/D5 region of KIT, e.g., human KIT, whereinthe antibody has higher affinity to a KIT antigen consisting essentiallyof the amino acid sequence of SEQ ID NO: 16 or 17, than to a KIT antigenconsisting essentially of the amino acid sequence of SEQ ID NO: 18 or19, as determined by methods known in the art, e.g., ELISA or Biacoreassay. In another specific embodiment, an antibody described herein (oran antigen-binding fragment thereof) immunospecifically binds a D4/D5region of KIT, e.g., human KIT, wherein the antibody has higher affinityto a KIT antigen consisting essentially of the amino acid sequence ofSEQ ID NO: 16 or 17, than to a KIT antigen consisting essentially of theamino acid sequence of SEQ ID NO: 18 or 19, as determined by methodsknown in the art, e.g., ELISA or Biacore assay, and wherein the higheraffinity is at least 2 fold, 5 fold, 10 fold, 50 fold, 100 fold, or 500fold.

In particular embodiments, an antibody described herein (or anantigen-binding fragment thereof) does not bind the extracellular ligandbinding site of KIT, e.g., the SCF binding site of KIT.

In particular embodiments, an antibody described herein (or anantigen-binding fragment thereof) does not inhibit ligand binding toKIT, e.g., does not inhibit KIT ligand (e.g., SCF) binding to KIT. Incertain embodiments, an antibody described herein does not block orinhibit KIT dimerization. In a particular embodiment, an antibodydescribed herein does not disrupt a KIT dimer (for example, does notinduce dissociation of a KIT dimer into KIT monomers). In particularembodiments, an antibody described herein does not inhibit KITdimerization and/or does not inhibit or block KIT ligand (e.g., SCF)binding to KIT (e.g., human KIT).

In certain embodiments, an antibody described herein blocks or inhibitsKIT dimerization or disrupts a KIT dimer (for example, inducesdissociation of a KIT dimer into KIT monomers). In particularembodiments, an antibody described herein inhibits KIT dimerization orinduces dissociation of a KIT dimer, and does not inhibit or block KITligand (e.g., SCF) binding to KIT (e.g., human KIT).

In specific aspects, antibodies described herein are inhibitoryantibodies, that is, antibodies that inhibit (e.g., partially inhibit)KIT activity, i.e., one or more KIT activities. In a specificembodiment, partial inhibition of a KIT activity results in, forexample, about 25% to about 65% or 75% inhibition. In a specificembodiment, partial inhibition of a KIT activity results in, forexample, about 35% to about 85% or 95% inhibition. Non-limiting examplesof KIT activities include KIT dimerization, KIT phosphorylation (e.g.,tyrosine phosphorylation), signaling downstream of KIT (e.g., Stat, AKT,MAPK, or Ras signaling), induction or enhancement of gene transcription(e.g., c-Myc), induction or enhancement of cell proliferation or cellsurvival. In a particular embodiment, an antibody described hereininhibits KIT phosphorylation. In a specific embodiment, an antibodydescribed herein inhibits KIT tyrosine phosphorylation in the KITcytoplasmic domain. In another particular embodiment, an antibodydescribed herein inhibits cell proliferation. In yet another particularembodiment, an antibody described herein inhibits cell survival. In aspecific embodiment, an antibody described herein induces apoptosis. Inanother specific embodiment, an antibody described herein induces celldifferentiation, e.g., cell differentiation in a cell expressing KIT,e.g., human KIT. In a particular embodiment, an antibody describedherein inhibits KIT activity but does not inhibit KIT dimerization. Inanother particular embodiment, an antibody described herein inhibits KITactivity and does not inhibit ligand binding to KIT, e.g., does notinhibit KIT ligand (e.g., SCF) binding to KIT, but does inhibit KITdimerization.

In a particular embodiment, an antibody described herein inhibits a KITactivity, such as ligand-induced tyrosine phosphorylation of a KITcytoplasmic domain, by about 25% to about 65% or 75%, as determined by acell-based phosphorylation assay well known in the art, for example, thecell-based phosphorylation assay described in Example 3 (Section 6.3)below. In a certain embodiment, an antibody described herein inhibits aKIT activity, such as ligand-induced tyrosine phosphorylation of a KITcytoplasmic domain, by about 35% to about 85% or 95%, as determined by acell-based phosphorylation assay well known in the art, for example, thecell-based phosphorylation assay described in Example 3 (Section 6.3)below. In a particular embodiment, an antibody described herein (e.g.,antibody 37M or 37C, an antigen-binding fragment thereof, an antibodycomprising CDRs of antibody 37M, or a conjugate comprising, for example,antibody 37M or 37C, a KIT-binding fragment thereof, or an antibodycomprising CDRs of antibody 37M, linked, covalently or noncovalently, toa therapeutic agent) inhibits a KIT activity, such as ligand-inducedtyrosine phosphorylation of a KIT cytoplasmic domain, with a 50%inhibition concentration (IC₅₀) of less than about 500 pM, or less thanabout 250 pM, as determined by a cell-based phosphorylation assay wellknown in the art, for example, the cell-based phosphorylation assaydescribed in Example 3 (Section 6.3) below. In a specific embodiment,the IC₅₀ is less than about 200 pM. In a specific embodiment, the IC₅₀is in the range of about 50 pM to about 225 pM, or in the range of 100pM to about 180 pM. In a specific embodiment, the IC₅₀ is in the rangeof about 50 pM to about 150 pM, or about 50 pM to about 125 pM, or about50 pM to about 110 pM.

In a specific embodiment, an antibody described herein (i)immunospecifically binds to a KIT polypeptide comprising the D4/D5region of human KIT, (ii) inhibits KIT phosphorylation (e.g., tyrosinephosphorylation), and (iii) does not affect KIT ligand (e.g., SCF)binding to KIT. In yet another specific embodiment, such an antibodydoes not inhibit KIT dimerization.

The antibodies provided herein generally do not immunospecifically bindto the D1, D2, or D3 domain of the extracellular domain of KIT, e.g.,human KIT. That is, in some embodiments, an antibody described hereindoes not immunospecifically bind to a D1 domain of the extracellulardomain of KIT (e.g., human KIT); in some embodiments, an antibodydescribed herein does not immunospecifically bind to a D2 domain of theextracellular domain of KIT (e.g., human KIT); and in some embodiments,an antibody described herein does not immunospecifically bind to a D3domain of the extracellular domain of KIT (e.g., human KIT). In someembodiments, an anti-KIT antibody described herein does notimmunospecifically binds to a D5 domain of the extracellular domain ofKIT (e.g., human KIT). In some embodiments, an antibody described hereindoes not specifically bind to a D4-D5 hinge region of KIT (e.g., humanKIT). In certain embodiments, an antibody described herein does notimmunospecifically bind to domain D1, D2, and/or D3 of KIT (e.g., humanKIT).

In other specific embodiments, an antibody described hereinimmunospecifically binds to a monomeric form of KIT (e.g., human KIT).In particular embodiments, an antibody described herein does notimmunospecifically bind to a monomeric form of KIT (e.g., human KIT). Inspecific embodiments, an antibody described herein immunospecificallybinds to a dimeric form of KIT (e.g., human KIT). In specificembodiments, an antibody described herein does not bind to a monomericform of KIT and specifically binds to a dimeric form of KIT ormultimeric form of KIT. In certain embodiments, an antibody has higheraffinity for a KIT monomer than a KIT dimer. In certain embodiments, anantibody has higher affinity for a KIT monomer than a KIT multimer.

In specific embodiments, an anti-KIT antibody described herein (or anantigen-binding fragment thereof) specifically binds to a native isoformor native variant of KIT (that is a naturally occurring isoform orvariant of KIT in an animal (e.g., monkey, mouse, goat, donkey, dog,cat, rabbit, pig, rat, human, frog, or bird) that can be isolated froman animal, preferably a human). In particular embodiments, an antibodydescribed herein immunospecifically binds to human KIT or a fragmentthereof. In specific embodiments, an anti-KIT antibody described hereinspecifically binds to human KIT or a fragment thereof and does notspecifically bind to a non-human KIT (e.g., monkey, mouse, goat, donkey,dog, cat, rabbit, pig, rat, or bird) or a fragment thereof. In specificembodiments, an anti-KIT antibody described herein specifically binds tohuman KIT or a fragment thereof and does not specifically bind to murineKIT. In certain embodiments, an anti-KIT antibody described hereinspecifically binds to human KIT or a fragment thereof and does notspecifically bind to dog (canine) KIT. In certain embodiments, anantibody described herein specifically binds to v-Kit or a fragmentthereof (see, e.g., Besmer et al., Nature, 1986, 320:415-21).

In some embodiments, an antibody described herein (or an antigen-bindingfragment thereof) immunospecifically binds to a KIT antigen comprisingone or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20) amino acidsubstitutions (e.g., conservative amino acid substitutions), deletions,or additions of the amino acid sequence depicted by SEQ ID NO: 14 or 15.In some embodiments, an antibody described herein immunospecificallybinds to a KIT antigen that has at least about 70%, 75%, 80%, 85%, 90%,95%, or 98% amino acid sequence identity to the amino acid sequencedepicted by SEQ ID NO: 14 or 15, for example, at least about 70%, 75%,80%, 85%, 90%, 95%, or 98% amino acid sequence identity to the aminoacid sequence depicted by SEQ ID NO: 14 or 15 over the entire length. Insome embodiments, an antibody described herein immunospecifically bindsto a KIT antigen comprising at most 10 amino acid substitutions,deletions, or additions; at most 8 amino acid substitutions, deletions,or additions; at most 7 amino acid substitutions, deletions, oradditions; at most 6 conservative amino acid substitutions, deletions,or additions; at most 5 amino acid substitutions, deletions, oradditions, at most 4 amino acid substitutions, deletions, or additions,at most 3 amino acid substitutions, deletions, or additions or at most 2amino acid substitutions, deletions, or additions of the amino acidsequence depicted by SEQ ID NO: 14 or 15.

In some embodiments, an antibody described herein (or an antigen-bindingfragment thereof) immunospecifically binds to a KIT antigen comprisingone or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) conservative aminoacid substitutions of the amino acid sequence depicted by SEQ ID NO: 14or 15. In some embodiments, an antibody described hereinimmunospecifically binds to a KIT antigen comprising at most 10conservative amino acid substitutions, at most 8 conservative amino acidsubstitutions, at most 7 conservative amino acid substitutions, at most6 conservative amino acid substitutions, or at most 5 conservative aminoacid substitutions of the amino acid sequence depicted by SEQ ID NO: 14or 15.

In certain embodiments, an antibody described herein does notimmunospecifically bind to a transmembrane region of KIT (e.g., humanKIT). In some embodiments, an antibody described herein does notimmunospecifically bind to a cytoplasmic juxtamembrane domain of KIT(e.g., human KIT). In some embodiments, an antibody described hereindoes not immunospecifically bind to a cytoplasmic domain of KIT (e.g.,human KIT). In certain embodiments, an antibody described herein doesnot immunospecifically bind to a split cytoplasmic kinase domain of KIT(e.g., human KIT). In certain embodiments, an antibody described hereindoes not immunospecifically bind to a tyrosine phosphorylated epitope ofKIT (e.g., human KIT).

In certain embodiments, an antibody described herein or antigen-bindingfragment thereof (e.g., antibody comprising VL CDR1, VL CDR2, and VLCDR3 having the amino acid sequence of SEQ ID NO: 20, 21, and 22,respectively, and/or a VH chain region comprising VH CDR1, VH CDR2, andVH CDR3 having the amino acid sequence of SEQ ID NO: 23, 24, and 25,respectively) binds to an extracellular domain of human KIT comprising amutation, for example a somatic mutation associated with cancer (e.g.,GIST), such as a mutation in exon 9 of human KIT wherein the Ala and Tyrresidues at positions 502 and 503 are duplicated. In certainembodiments, an antibody described herein or antigen-binding fragmentthereof (e.g., antibody comprising VL CDR1, VL CDR2, and VL CDR3 havingthe amino acid sequence of SEQ ID NO: 20, 21, and 22, respectively,and/or a VH chain region comprising VH CDR1, VH CDR2, and VH CDR3 havingthe amino acid sequence of SEQ ID NO: 23, 24, and 25, respectively)binds to an extracellular domain of wild-type human KIT and anextracellular domain of human KIT comprising a mutation, for example asomatic mutation associated with cancer (e.g., GIST), such as a mutationin exon 9 of human KIT wherein the Ala and Tyr residues at positions 502and 503 are duplicated (see, e.g., Marcia et al., (2000) Am. J. Pathol.156(3):791-795; and Debiec-Rychter et al., (2004) European Journal ofCancer. 40:689-695, which are both incorporated herein by reference intheir entireties, describing KIT mutations).

In certain embodiments, an antibody described herein or antigen-bindingfragment thereof (e.g., antibody comprising VL CDR1, VL CDR2, and VLCDR3 having the amino acid sequence of SEQ ID NO: 20, 21, and 22,respectively, and/or a VH chain region comprising VH CDR1, VH CDR2, andVH CDR3 having the amino acid sequence of SEQ ID NO: 23, 24, and 25,respectively) binds to an extracellular domain of human KIT which isglycosylated. In certain embodiments, an antibody described herein orantigen-binding fragment thereof (e.g., antibody comprising VL CDR1, VLCDR2, and VL CDR3 having the amino acid sequence of SEQ ID NO: 20, 21,and 22, respectively, and/or a VH chain region comprising VH CDR1, VHCDR2, and VH CDR3 having the amino acid sequence of SEQ ID NO: 23, 24,and 25, respectively) binds to two different glycosylated forms of anextracellular domain of human KIT. For example, two forms of human KITwith different molecular weights, indicating different glycosylationpatterns, have been observed by immunoblotting. In certain embodiments,an antibody described herein may specifically bind to both of theseforms of human KIT which have different glycosylation patterns, e.g.,one form is more glycosylated than the other. In certain embodiments, anantibody described herein or antigen-binding fragment thereof (e.g.,antibody comprising VL CDR1, VL CDR2, and VL CDR3 having the amino acidsequence of SEQ ID NO: 20, 21, and 22, respectively, and/or a VH chainregion comprising VH CDR1, VH CDR2, and VH CDR3 having the amino acidsequence of SEQ ID NO: 23, 24, and 25, respectively) binds to anextracellular domain of human KIT which is not glycosylated.

In a particular embodiment, an antibody described herein is not anantibody described by International Patent Application No. WO2008/153926, the contents of which are incorporated herein by referencein its entirety. In another particular embodiment, an antibody describedherein does not immunospecifically bind to a KIT epitope described byInternational Patent Application No. WO 2008/153926, for example anepitope consisting essentially of the amino acid sequence

(SEQ ID NO: 38) SELHLTRLKGTEGGTYT or (SEQ ID NO: 39) LTRLKGTEGG.

In certain embodiments, an anti-KIT antibody described herein is not anantibody selected from the group consisting of: SR-1 antibody (see U.S.Patent Application Publication No. US 2007/0253951 A1; InternationalPatent Application Publication No. WO 2007/127317); anti-KIT antibodyobtained from hybridoma cell lines DSM ACC 2007, DSM ACC 2008, or DSMACC 2009, which have been deposited at the Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH, DSM, Mascheroder Weg 1 b, D-38124Braumschweig, Germany (see U.S. Pat. No. 5,545,533; International PatentApplication Publication No. WO 92/021766); antibody produced byhybridoma cell line DSM ACC 2247 (or A3C6E2; Deposit No. DSM ACC 2247,at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSM,Mascheroder Weg 1 b, D-38124 Braumschweig, Germany) (see U.S. Pat. No.5,808,002); and anti-KIT antibodies designated K27, K44, K45, K49, K57,K69, and K94 (see, e.g., Blechman et al., Stem Cells, 1993, 11:12-21;Blechman et al., Cell, 1995, 80:103-113; Lev et al., Mol. Cell. Biol.,1993, 13:2224-2234; and European Patent Application Publication No.EP0548867 A2). In certain embodiments, an anti-KIT antibody describedherein does not comprise a CDR of an antibody selected from such group.In particular embodiments, an anti-KIT antibody described herein doesnot comprise one or more (e.g., two, three, four, five, or six) CDRs(e.g., 3 VL CDRs and/or 3 VH CDRs) of an antibody selected from suchgroup. In another embodiment, an antibody described herein is notcompetitively blocked (e.g., competitively blocked in a dose-dependentmanner) by one of those antibodies, for example, as determined bycompetition binding assays (e.g., ELISAs). In certain embodiments, ananti-KIT antibody described herein is not antibody Ab1 or Ab21, which isdescribed in U.S. Provisional Application No. 61/426,387, filed Dec. 22,2010. In certain embodiments, an anti-KIT antibody described herein isnot an antibody selected from the group consisting of: Ab1, Ab2, Ab3,Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16,Ab17, Ab18, Ab19, Ab20, and Ab21, as described in U.S. ProvisionalApplication No. 61/426,387, filed Dec. 22, 2010 and Ab24-Ab192 asdescribed in PCT International Patent Application No. PCT/US2011/29980filed Mar. 25, 2011. In certain embodiments, an anti-KIT antibodydescribed herein does not comprise a CDR, or one or more CDRs (e.g., 3VL CDRs and/or 3VH CDRs), of an antibody selected from the groupconsisting of: Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11,Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, and Ab21, asdescribed in U.S. Provisional Application No. 61/426,387, filed Dec. 22,2010, and Ab24-Ab192 as described in PCT International PatentApplication No. PCT/US2011/29980 filed Mar. 25, 2011. In particularembodiments, an anti-KIT antibody described herein does not comprise aCDR, or one or more CDRs (e.g., 3 VL CDRs and/or 3VH CDRs), VL chainregion, or VH chain region of an antibody selected from the antibodies(e.g., antibodies Ab1-Ab21 and Ab24-Ab192) described in U.S. ProvisionalApplication No. 61/426,387 filed Dec. 22, 2010 or PCT InternationalPatent Application No. PCT/US2011/29980 filed Mar. 25, 2011. In certainembodiments, an anti-KIT antibody described herein is not antibody Ab1or Ab21, or an antibody comprising CDRs (e.g., one, two, three, four,five, or six CDRs) of antibody Ab1 or Ab21, as described in U.S.Provisional Application No. 61/426,387, filed Dec. 22, 2010.

In a particular embodiment, an antibody described herein orantigen-binding fragment thereof, which immunospecifically bind to a KITpolypeptide (e.g., the D4/D5 region of KIT, for example, human KIT),does not comprise one or more (e.g., two, three, four, five, or six)CDRs (e.g., VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3) ofan antibody described in US Patent Application Publication NO. US2008/0287309, for example antibody 36C1, 84H7, 63C10, or 65A12.

In a specific embodiment, an antibody described herein is not anantibody produced by the hybridoma (BA7.3C.9) having the American TypeCulture Collection (ATCC) Accession number HB10716, as described forexample in U.S. Pat. No. 5,919,911 or 5,489,516. In another specificembodiment, an antibody described herein does not comprise the CDRs(e.g., VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and/or VH CDR3) ofthe antibody produced by the hybridoma (BA7.3C.9) having the AmericanType Culture Collection (ATCC) Accession number HB10716, as describedfor example in U.S. Pat. No. 5,919,911 or 5,489,516. In another specificembodiment, an antibody described herein does not comprise the CDRs ofthe SR-1 antibody described for example in U.S. Pat. No. 5,919,911 or5,489,516 or U.S. Patent Application Publication No. US 2007/0253951 A1(see, e.g., ¶ [0032] or ¶ [0023]). In a further embodiment, an antibodydescribed herein is not a humanized antibody of the antibody produced bythe hybridoma (BA7.3C.9) having the American Type Culture Collection(ATCC) Accession number HB10716, as described for example in U.S. Pat.No. 5,919,911 or 5,489,516.

In a specific embodiment, an antibody described herein is not thehumanized antibodies of the SR-1 antibody as described in U.S. PatentApplication Publication No. US 2007/0253951 A1. In a specificembodiment, an antibody described herein does not comprise one or moreamino acid sequences selected from the group consisting of: SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ ID NO: 10referenced in U.S. Patent Application Publication No. US 2007/0253951A1. In a particular embodiment, an antibody described herein does notcomprise the amino acid sequences of SEQ ID NOs: 2 and 4 or of SEQ IDNOs: 2 and 6 referenced in U.S. Patent Application Publication No. US2007/0253951 A1. In a specific embodiment, an antibody described hereindoes not comprise one or more amino acid sequence that is at least 90%identical to the amino acid sequence selected from the group consistingof: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, and SEQ IDNO: 10 referenced in U.S. Patent Application Publication No. US2007/0253951 A1. In a particular embodiment, an antibody describedherein does not comprise one or more CDRs described in U.S. PatentApplication Publication No. US 2007/0253951 A1, for example, amino acids44 to 58 of SEQ ID NO: 8 (VL CDR1 of antibody SR-1; RASESVDIYGNSFMH),amino acids 74 to 80 of SEQ ID NO: 8 (VL CDR2 of antibody SR-1;LASNLES), amino acids 111 to 121 of SEQ ID NO: 8 (VL CDR3 of antibodySR-1; QQNNEDPYT), amino acids 50 to 54 of SEQ ID NO: 10 (VH CDR1 ofantibody SR-1; SYNMH), amino acids 69 to 85 of SEQ ID NO: 10 (VH CDR2 ofantibody SR-1; VIYSGNGDTSYNQKFKG), and/or amino acids 118 to 125 of SEQID NO: 10 (VH CDR3 of antibody SR-1; RDTRFGN), where SEQ ID NOs: 8 and10 are those referenced in U.S. Patent Application Publication No. US2007/0253951 A1 (see, e.g., ¶ [0032] or ¶ [0023]). In a particularembodiment, an antibody described herein does not comprise one or moreCDRs described in U.S. Patent Application Publication No. US2007/0253951 A1, for example, amino acids 43 to 58 of SEQ ID NO: 2 (VLCDR1), amino acids 74 to 80 of SEQ ID NO: 2 (VL CDR2), amino acids 113to 121 of SEQ ID NO: 2 (VL CDR3), amino acids 50 to 54 of SEQ ID NO: 4(VH CDR1), amino acids 69 to 85 of SEQ ID NO: 4 (VH CDR2), and/or aminoacids 118 to 125 of SEQ ID NO: 4 (VH CDR3), where SEQ ID NOs: 2 and 4are those referenced in U.S. Patent Application Publication No. US2007/0253951 A1. In a particular embodiment, an antibody describedherein is not a humanized antibody of antibody SR-1 as described in U.S.Patent Application Publication No. US 2007/0253951 A1.

In a specific embodiment, an antibody described herein is not anantibody selected from the group consisting of: antibody Anti-S100,ACK2, and ACK4 as described in U.S. Pat. No. 6,989,248 or 7,449,309. Ina specific embodiment, an antibody described herein does not compriseone or more CDRs (e.g., 3 VL CDRs and/or 3 VH CDRs) of an antibodyselected from the group consisting of: antibody Anti-S100, ACK2, andACK4 as described in U.S. Pat. No. 6,989,248 or 7,449,309.

In particular aspects, provided herein are antibodies (e.g., a murine,chimeric or humanized antibody) or an antigen-binding fragment thereof,which immunospecifically bind to a KIT polypeptide (e.g., the D4/D5region of KIT, for example, human KIT) and comprise an amino acidsequence as described herein, as well as antibodies which compete (e.g.,compete in a dose-dependent manner) with such antibodies for binding toa KIT polypeptide.

In a particular embodiment, an antibody described herein, or anantigen-binding fragment thereof, comprises a VL chain region having theamino acid sequence of SEQ ID NO: 2, and/or comprises a VH chain regionhaving the amino acid sequence of SEQ ID NO: 3 or 5. In a particularembodiment, an antibody described herein comprises a VL chain regionhaving the amino acid sequence of SEQ ID NO: 2, and comprises a VH chainregion having the amino acid sequence of SEQ ID NO: 3. In a particularembodiment, an antibody described herein comprises a VL chain regionhaving the amino acid sequence of SEQ ID NO: 2, and comprises a VH chainregion having the amino acid sequence of SEQ ID NO: 5. In certainembodiments, any of these antibodies can comprise a variable heavy chainand a variable light chain, for example, a separate variable heavy chainand a separate variable light chain. In a specific embodiment, theposition (i.e., boundary) of a VL chain region described herein relativeto the constant region may change by one, two, three, or four amino acidpositions so long as immunospecific binding to KIT (e.g., the D4/D5region of human KIT) is maintained (e.g., substantially maintained, forexample, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%). In a specific embodiment, the position (i.e.,boundary) of a VH chain region described herein relative to the constantregion may change by one, two, three, or four amino acid positions solong as immunospecific binding to KIT (e.g., the D4/D5 region of humanKIT) is maintained (e.g., substantially maintained, for example, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%).

In certain embodiments, an antibody described herein, or anantigen-binding fragment thereof, comprises a variable light (VL) chainregion comprising an amino acid sequence described herein (e.g., seeFIG. 3A). In certain embodiments, an antibody described herein comprisesa VL chain region having the amino acid sequence of SEQ ID NO: 2,wherein the antibody immunospecifically binds to a KIT polypeptide,e.g., a human KIT polypeptide, for example, a D4/D5 region of KIT (e.g.,human KIT), for example SEQ ID NO: 15. In certain embodiments, any ofthese antibodies can comprise a variable variable light chain, forexample, a separate variable light chain. In certain embodiments, anantibody described herein comprises a VL chain region having the aminoacid sequence of SEQ ID NO: 2.

In certain embodiments, an antibody described herein, or anantigen-binding fragment thereof, comprises a variable heavy (VH) chainregion comprising an amino acid sequence described herein (e.g., seeFIG. 3B). In certain embodiments, an antibody described herein comprisesa VH chain region having the amino acid sequence of SEQ ID NO: 3 or 5,wherein the antibody immunospecifically binds to a KIT polypeptide,e.g., a human KIT polypeptide, for example, a D4/D5 region of KIT (e.g.,human KIT), for example SEQ ID NO: 15. In certain embodiments, anantibody described herein comprises a VH chain region having the aminoacid sequence of SEQ ID NO: 3. In certain embodiments, an antibodydescribed herein comprises a VH chain region having the amino acidsequence of SEQ ID NO: 5. In certain embodiments, any of theseantibodies can comprise a variable heavy chain, for example, a separatevariable heavy chain.

In certain embodiments, an antibody described herein, or anantigen-binding fragment thereof, comprises one or more VL CDRs havingthe amino acid sequence described herein (e.g., see Tables 1), whereinthe antibody immunospecifically binds to a KIT polypeptide, e.g., ahuman KIT polypeptide, for example, a D4/D5 region of KIT (e.g., humanKIT), for example SEQ ID NO: 15. In certain embodiments, an antibodydescribed herein comprises at least two VL CDRs from the same antibodysequence depicted at Tables 1, e.g., comprises VL CDR1 and VL CDR2 ofantibody 37C. In certain embodiments, an antibody described hereincomprises a VL CDR1, VL CDR2, and VL CDR3 from the same antibodysequence depicted at Tables 1, e.g., comprises VL CDR1, VL CDR2, and VLCDR3 of antibody 37C.

In particular embodiments, the VL CDR1 has the amino acid sequence ofSEQ ID NO: 20. In certain embodiments, the VL CDR2 has the amino acidsequence of SEQ ID NO: 21. In specific embodiments, the VL CDR3 has theamino acid sequence of SEQ ID NO: 22. In a specific embodiment, anantibody described herein comprises a VL chain region comprising (i) aVL CDR1 having the amino acid sequence of SEQ ID NO: 20; (ii) a VL CDR2having the amino acid sequence of SEQ ID NO: 21; and (iii) a VL CDR3having the amino acid sequence of SEQ ID NO: 22, wherein the antibodyimmunospecifically binds to a KIT polypeptide, e.g., a human KITpolypeptide, for example, a D4/D5 region of KIT (e.g., human KIT), forexample SEQ ID NO: 15. In a specific embodiment, a VL CDR1 has thesequence of SEQ ID NO: 20 that does not have an amino acid substitutionat position 7 of SEQ ID NO: 20 (for example, the R amino acid atposition 7 of SEQ ID NO: 20 is not substituted with G). In a specificembodiment, a VL CDR3 has the sequence of SEQ ID NO: 22 that does nothave an amino acid substitution at position 8 of SEQ ID NO: 22 (forexample, the R amino acid at position 8 of SEQ ID NO: 22 is notsubstituted with L).

In certain embodiments, an antibody described herein, or anantigen-binding fragment thereof, comprises one or more VH CDRs havingthe amino acid sequence described herein (e.g., see Table 3), whereinthe antibody immunospecifically binds to a KIT polypeptide, e.g., ahuman KIT polypeptide, for example, a D4/D5 region of KIT (e.g., humanKIT), for example SEQ ID NO: 15. In particular embodiments, the VH CDR1has the amino acid sequence of SEQ ID NO: 23. In certain embodiments,the VH CDR2 has the amino acid sequence of SEQ ID NO: 24. In specificembodiments, the VH CDR3 has the amino acid sequence of SEQ ID NO: 25.In certain embodiments, an antibody described herein comprises at leasttwo VH CDRs from the same antibody sequence depicted at Table 3, e.g.,comprises VH CDR1 and VH CDR2 of antibody 37 M or 37C. In certainembodiments, an antibody described herein comprises a VH CDR1, VH CDR2,and VH CDR3 from the same antibody sequence depicted at Table 3, e.g.,comprises VH CDR1, VH CDR2, and VH CDR3 of antibody 37M or 37C.

In a specific embodiment, an antibody described herein, or anantigen-binding fragment thereof, comprises a VH chain region comprising(i) a VH CDR1 having the amino acid sequence of SEQ ID NO: 23; (ii) a VHCDR2 having the amino acid sequence of SEQ ID NO: 24; and (iii) a VHCDR3 having the amino acid sequence of SEQ ID NO: 25, wherein theantibody immunospecifically binds to a KIT polypeptide, e.g., a humanKIT polypeptide, for example, a D4/D5 region of KIT (e.g., human KIT),for example SEQ ID NO: 15.

In another specific embodiment, an antibody (e.g., murine, chimeric orhumanized antibody) described herein, or an antigen-binding fragmentthereof, comprises (i) a VL chain region comprising a VL CDR1, VL CDR2,and VL CDR3 having the amino acid sequences of SEQ ID NO: 20, 21, and22, respectively; and (ii) a VH chain region comprising a VH CDR1, VHCDR2, and VH CDR3 having the amino acid sequences of SEQ ID NO: 23, 24,and 25, respectively, wherein the antibody immunospecifically binds to aKIT polypeptide, e.g., a human KIT polypeptide, for example, a D4/D5region of KIT (e.g., human KIT), for example SEQ ID NO: 15.

In another specific embodiment, an antibody (e.g., murine, chimeric orhumanized antibody) described herein, or an antigen-binding fragmentthereof, comprises (i) a VL chain region comprising a VL CDR1, VL CDR2,and VL CDR3 having the amino acid sequences of SEQ ID NO: 20, 21, and22, respectively; and (ii) a VH chain region having the amino acidsequence of SEQ ID NO: 3 or 5, wherein the antibody immunospecificallybinds to a KIT polypeptide, e.g., a human KIT polypeptide, for example,a D4/D5 region of KIT (e.g., human KIT), for example SEQ ID NO: 15.

In another specific embodiment, an antibody (e.g., murine, chimeric orhumanized antibody) described herein, or an antigen-binding fragmentthereof, comprises (i) a VL chain region having the amino acid sequenceof SEQ ID NO: 2; and (ii) a VH chain region comprising a VH CDR1, VHCDR2, and VH CDR3 having the amino acid sequences of SEQ ID NO: 23, 24,or 25, respectively, wherein the antibody immunospecifically binds to aKIT polypeptide, e.g., a human KIT polypeptide, for example, a D4/D5region of KIT (e.g., human KIT), for example SEQ ID NO: 15.

Also provided herein are antibodies that bind the same or an overlappingepitope as an antibody described herein (e.g., antibody 37M or 37C),i.e., antibodies that compete for binding to KIT, or bind epitopes whichoverlap with epitopes bound by the antibodies described herein, e.g., anepitope located on a D4/D5 region of human KIT. Antibodies thatrecognize such epitopes can be identified using routine techniques suchas an immunoassay, for example, by showing the ability of one antibodyto block the binding of another antibody to a target antigen, i.e., acompetitive binding assay. Competition binding assays also can be usedto determine whether two antibodies have similar binding specificity foran epitope. Competitive binding can be determined in an assay in whichthe immunoglobulin under test inhibits specific binding of a referenceantibody to a common antigen, such as KIT. Numerous types of competitivebinding assays are known, for example: solid phase direct or indirectradioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay (see Stahli et al., (1983)Methods in Enzymology 9:242); solid phase direct biotin-avidin EIA (seeKirkland et al., (1986) J. Immunol. 137:3614); solid phase directlabeled assay, solid phase direct labeled sandwich assay (see Harlow andLane, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press);solid phase direct label RIA using I-125 label (see Morel et al., (1988)Mol. Immunol. 25(1):7); solid phase direct biotin-avidin EIA (Cheung etal., (1990) Virology 176:546); and direct labeled RIA. (Moldenhauer etal., (1990) Scand J. Immunol. 32:77). Typically, such an assay involvesthe use of purified antigen (e.g., KIT, such as extracellular domain ofKIT or a D4/D5 region of KIT) bound to a solid surface or cells bearingeither of these, an unlabeled test immunoglobulin and a labeledreference immunoglobulin. Competitive inhibition can be measured bydetermining the amount of label bound to the solid surface or cells inthe presence of the test immunoglobulin. Usually the test immunoglobulinis present in excess. Usually, when a competing antibody is present inexcess, it will inhibit specific binding of a reference antibody to acommon antigen by at least 50-55%, 55-60%, 60-65%, 65-70% 70-75% ormore. A competition binding assay can be configured in a large number ofdifferent formats using either labeled antigen or labeled antibody. In acommon version of this assay, the antigen is immobilized on a 96-wellplate. The ability of unlabeled antibodies to block the binding oflabeled antibodies to the antigen is then measured using radioactive orenzyme labels. For further details see, for example, Wagener et al., J.Immunol., 1983, 130:2308-2315; Wagener et al., J. Immunol. Methods,1984, 68:269-274; Kuroki et al., Cancer Res., 1990, 50:4872-4879; Kurokiet al., Immunol. Invest., 1992, 21:523-538; Kuroki et al., Hybridoma,1992, 11:391-407, and Using Antibodies: A Laboratory Manual, Ed Harlowand David Lane editors (Cold Springs Harbor Laboratory Press, ColdSprings Harbor, N.Y., 1999), pp. 386-389.

In certain aspects, competition binding assays can be used to determinewhether an antibody is competitively blocked, e.g., in a dose dependentmanner, by another antibody for example, an antibody binds essentiallythe same epitope, or overlapping epitopes, as a reference antibody, whenthe two antibodies recognize identical or sterically overlappingepitopes in competition binding assays such as competition ELISA assays,which can be configured in all number of different formats, using eitherlabeled antigen or labeled antibody. In a particular embodiment, anantibody can be tested in competition binding assays with an antibodydescribed herein, e.g., antibody 37M or 37C, an antibody comprising VHCDRs and VL CDRs of 37M or 37C, or a humanized monoclonal antibodycomprising VH CDRs and VL CDRs of antibody 37M or 37C. In a specificembodiment, a competition binding assay is carried out, for example, asdescribed in section 6.6 below.

In specific aspects, provided herein is an antibody which competitivelyblocks (e.g., in a dose dependent manner), antibodies comprising theamino acid sequences described herein for specific binding to a KITpolypeptide (e.g., a D4/D5 region of KIT, for example human KIT), asdetermined using assays known to one of skill in the art or describedherein (e.g., ELISA competitive assays). In particular embodiments, suchcompetitively blocking antibody inhibits one or more KIT activities. Inspecific aspects, provided herein is an antibody which competes (e.g.,in a dose dependent manner) for specific binding to a KIT polypeptide(e.g., a D4/D5 region of KIT, for example human KIT), with an antibodycomprising the amino acid sequences described herein, as determinedusing assays known to one of skill in the art or described herein (e.g.,ELISA competitive assays). In a certain embodiment, antibody 37M is anexample of an antibody which competes, competitively inhibits (e.g., ina dose-dependent manner) or competitively blocks, antibody 37C frombinding to a KIT polypeptide (e.g., a D4/D5 region of KIT, for examplehuman KIT). In a particular embodiment, antibody 37C is an example of anantibody which competes, competitively inhibits, or competitively blocks(e.g., in a dose-dependent manner) antibody 37M from binding to a KITpolypeptide (e.g., a D4/D5 region of KIT, for example human KIT).

In specific aspects, provided herein is an antibody which competes(e.g., in a dose dependent manner) for specific binding to a KITpolypeptide (e.g., a D4/D5 region of KIT, for example human KIT), withan antibody comprising a VL chain region having the amino acid sequenceof SEQ ID NO: 2 and a VH chain region having the amino acid sequence ofSEQ ID NO: 3 or 5.

In specific aspects, provided herein is an antibody which competes(e.g., in a dose dependent manner) for specific binding to a KITpolypeptide (e.g., a D4/D5 region of KIT, for example human KIT), withan antibody comprising a light chain comprising the amino acid sequenceof SEQ ID NO: 6 (or the amino acid sequence of SEQ ID NO: 6 starting atposition 20, lacking the signal peptide) and a heavy chain comprisingthe amino acid sequence of SEQ ID NO: 7 (or the amino acid sequence ofSEQ ID NO: 7 starting at position 20, lacking the signal peptide), forspecific binding to a KIT polypeptide (e.g., a D4/D5 region of KIT, forexample human KIT).

In specific aspects, provided herein is an antibody which competes(e.g., in a dose dependent manner) for specific binding to a KITpolypeptide (e.g., a D4/D5 region of KIT, for example human KIT), withan antibody comprising i) a VL chain region comprising a VL CDR1, VLCDR2, and VL CDR3 having the amino acid sequences of SEQ ID NO: 20, 21,and 22, respectively; and (ii) a VH chain region comprising a VH CDR1,VH CDR2, and VH CDR3 having the amino acid sequences of SEQ ID NO: 23,24, and 25, respectively.

In a specific embodiment, an antibody described herein is one that iscompetitively blocked (e.g., in a dose dependent manner) by an antibodycomprising a VL chain region having the amino acid sequence of SEQ IDNO: 2 and a VH chain region having the amino acid sequence of SEQ ID NO:3 or 5, for specific binding to a KIT polypeptide (e.g., a D4/D5 regionof KIT, for example human KIT).

In a specific embodiment, an antibody described herein is one that iscompetitively blocked (e.g., in a dose dependent manner) by an antibodycomprising a light chain comprising the amino acid sequence of SEQ IDNO: 6 (or the amino acid sequence of SEQ ID NO: 6 starting at position20, lacking the signal peptide) and a heavy chain comprising the aminoacid sequence of SEQ ID NO: 7 (or the amino acid sequence of SEQ ID NO:7 starting at position 20, lacking the signal peptide), for specificbinding to a KIT polypeptide (e.g., a D4/D5 region of KIT, for examplehuman KIT).

In another specific embodiment, an antibody described herein is one thatis competitively blocked (e.g., in a dose dependent manner) by anantibody comprising (i) a VL chain region comprising a VL CDR1, VL CDR2,and VL CDR3 having the amino acid sequences of SEQ ID NO: 20, 21, and22, respectively; and (ii) a VH chain region comprising a VH CDR1, VHCDR2, and VH CDR3 having the amino acid sequences of SEQ ID NO: 23, 24,and 25, respectively.

In another specific embodiment, such antibody is one that is notcompetitively blocked (e.g., in a dose dependent manner) by an antibodyprovided herein.

In another specific embodiment, an antibody described herein is one thatis not competitively blocked (e.g., in a dose dependent manner) byantibody Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12,Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, or Ab21 as described inU.S. Provisional Application No. 61/426,387, filed Dec. 22, 2010, or byan antibody comprising the CDRs of such antibodies. In another specificembodiment, an antibody described herein is one that is notcompetitively blocked (e.g., in a dose dependent manner) by an antibodycomprising (i) a VL chain region having the amino acid sequence:

DIQMTQSPTSLSAFVGDRVTITCQASQDIGNYLNWYQQKSGEPPKLLVYDASFLKKGVPSRFSGSGSGTQYFLTIYSLQPEDFATYFCQHSDNLSVTFGG  GTKVEVK(SEQ ID NO: 45; VL chain region of Ab1)  orDIQMTQSPTSLSAFVGDRVTITCQASQDIGNYLNWYQQKSGEPPKLLVYDASFLKKGVPSRFSGSGSGTQYFLTIYSLQPEDFATYFCQHSDSLSVTFGG GTKVEVK(SEQ ID NO: 47; VL chain region of Ab21); and(ii) a VH chain region having the amino acid  sequenceEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYLMSWVRQAPGKGLEWVSSIVPSGGFTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLQTGSWRVHAFDIWGQGTMVTVSS  (SEQ ID NO: 46; VH chain region of Ab1 or Ab21).

In another specific embodiment, an antibody described herein, whichcompetitively blocks (e.g., in a dose dependent manner) antibody 37M or37C (e.g., an antibody comprising (i) a VL chain region having the aminoacid sequence of SEQ ID NO: 2 and (ii) a VH chain region having theamino acid sequence of SEQ ID NO: 3 or 5), for specific binding to a KITpolypeptide (e.g., a D4/D5 region of KIT, for example human KIT), orbinds to the same epitope as antibody 37M or 37C, is not one of theantibodies selected from the group consisting of antibodies Ab1, Ab2,Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, Ab14, Ab15,Ab16, Ab17, Ab18, Ab19, Ab20, and Ab21 as described in U.S. ProvisionalApplication No. 61/426,387, filed Dec. 22, 2010, and an antibodycomprising CDRs of antibody Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9,Ab10, Ab11, Ab12, Ab13, Ab14, Ab15, Ab16, Ab17, Ab18, Ab19, Ab20, orAb21. In another specific embodiment, an antibody described herein,which competitively blocks (e.g., in a dose dependent manner) antibody37M or 37C (e.g., an antibody comprising (i) a VL chain region havingthe amino acid sequence of SEQ ID NO: 2 and (ii) a VH chain regionhaving the amino acid sequence of SEQ ID NO: 3 or 5), for specificbinding to a KIT polypeptide (e.g., a D4/D5 region of KIT, for examplehuman KIT), or binds to the same epitope as antibody 37M or 37C, is notone of the antibodies selected from the group consisting of antibodiesAb1-Ab21 as described in U.S. Provisional Application No. 61/426,387,filed Dec. 22, 2010, Antibodies Ab1-Ab21 and Ab24-Ab192 as described inPCT International Patent Application No. PCT/US2011/29980 filed Mar. 25,2011, and an antibody comprising CDRs of any one of antibodies Ab1-Ab21and Ab24-Ab192.

In another specific embodiment, an antibody described herein, whichcompetitively blocks (e.g., in a dose dependent manner) antibody 37M or37C (e.g., an antibody comprising (i) a VL chain region having the aminoacid sequence of SEQ ID NO: 2 and (ii) a VH chain region having theamino acid sequence of SEQ ID NO: 3 or 5), for specific binding to a KITpolypeptide (e.g., a D4/D5 region of KIT, for example human KIT), orbinds to the same epitope as antibody 37M or 37C, is not an antibodydescribed in PCT International Patent Application Publication No. WO2008/153926.

In another specific embodiment, an antibody described herein, whichcompetitively blocks (e.g., in a dose dependent manner) antibody 37M or37C (e.g., an antibody comprising (i) a VL chain region having the aminoacid sequence of SEQ ID NO: 2 and (ii) a VH chain region having theamino acid sequence of SEQ ID NO: 3 or 5), for specific binding to a KITpolypeptide (e.g., a D4/D5 region of KIT, for example human KIT), orbinds to the same epitope as antibody 37M or 37C, is not an antibodyselected from the group consisting of: (i) SR-1 antibody described inU.S. Patent Application Publication No. US 2007/0253951 A1 and/or PCTInternational Patent Application Publication No. WO 2007/127317); (ii)an antibody obtained from hybridoma cell line DSM ACC 2007, DSM ACC2008, or DSM ACC 2009, which have been deposited at the DeutscheSammlung von Mikroorganismen und Zellkulturen GmbH, DSM, Mascheroder Weg1 b, D-38124 Braumschweig, Germany (as described, e.g., in U.S. Pat. No.5,545,533; PCT International Patent Application Publication No. WO92/021766); (iii) an antibody produced by hybridoma cell line DSM ACC2247 (or A3C6E2; Deposit No. DSM ACC 2247, at the Deutsche Sammlung vonMikroorganismen und Zellkulturen GmbH, DSM, Mascheroder Weg 1 b, D-38124Braumschweig, Germany) as described, e.g., in U.S. Pat. No. 5,808,002;(iv) antibody designated K27, K44, K45, K49, K57, K69, or K94 asdescribed, e.g., in Blechman et al., Stem Cells, 1993, 11:12-21;Blechman et al., Cell, 1995, 80:103-113; Lev et al., Mol. Cell. Biol.,1993, 13:2224-2234; and European Patent Application Publication No.EP0548867 A2; and (v) an antibody comprising CDRs of any one of theantibodies described in (i)-(iv).

In specific aspects, provided herein is an antibody, or anantigen-binding fragment thereof, which immunospecifically binds to thesame epitope as that of an antibody comprising the amino acid sequencesdescribed herein for specific binding to a KIT polypeptide (e.g., aD4/D5 region of KIT, for example human KIT). Assays known to one ofskill in the art or described herein (e.g., ELISA assays) can be used todetermine if two antibodies bind to the same epitope.

In a specific embodiment, an antibody described herein, or anantigen-binding fragment thereof, immunospecifically binds to the sameepitope as that of an antibody comprising a VL chain region having theamino acid sequence of SEQ ID NO: 2 and a VH chain region having theamino acid sequence of SEQ ID NO: 3 or 5. In a specific embodiment, anantibody described herein, or an antigen-binding fragment thereof,immunospecifically binds to the same epitope as that of an antibodycomprising a light chain comprising the amino acid sequence of SEQ IDNO: 6 (or the amino acid sequence of SEQ ID NO: 6 starting at position20, lacking the signal peptide) and a heavy chain comprising the aminoacid sequence of SEQ ID NO: 7 (or the amino acid sequence of SEQ ID NO:7 starting at position 20, lacking the signal peptide).

In another specific embodiment, an antibody described herein, or anantigen-binding fragment thereof, immunospecifically binds to the sameepitope as that of an antibody comprising (i) a VL chain regioncomprising a VL CDR1, VL CDR2, and VL CDR3 having the amino acidsequences of SEQ ID NO: 20, 21, and 22, respectively; and (ii) a VHchain region comprising a VH CDR1, VH CDR2, and VH CDR3 having the aminoacid sequences of SEQ ID NO: 23, 24, and 25, respectively.

Table 1, below, presents the VL CDR (in particular, VL CDR1, VL CDR2,and VL CDR3) amino acid sequences of antibodies 37M and 37C. Table 2,below, presents the VL framework (FR) amino acid sequences (inparticular, VL FR1, VL FR2, VL FR3, and VL FR4 sequences) of antibodies37M and 37C. Table 3, below, presents the VH CDR (in particular, VHCDR1, VH CDR2, and VH CDR3) amino acid sequences of antibodies 37M and37C. Table 4, below, presents the VH FR amino acid sequences (inparticular, VH FR1, VH FR2, VH FR3, and VH FR4 sequences) of antibodies37M and 37C.

In certain aspects, an antibody described herein whichimmunospecifically binds to a D4/D5 region of a KIT polypeptide (e.g.,human KIT polypeptide) may be described by its VL chain region or VHchain region, or by its 3 VL CDRs or its 3 VH CDRs. See, for example,Rader et al., 1998, Proc. Natl. Acad. Sci. USA, 95: 8910-8915, which isincorporated herein by reference in its entirety, which describes thehumanization of the mouse anti-αvβ3 antibody by identifying acomplementing light chain or heavy chain, respectively, from a humanlight chain or heavy chain library, resulting in humanized antibodyvariants having affinities as high or higher than the affinity of theoriginal antibody. See also, Clackson et al., 1991, Nature, 352:624-628,which is incorporated herein by reference in its entirety, describingmethods of producing antibodies that bind a specific antigen by using aspecific VL (or VH) and screening a library for the complimentaryvariable domains. The screen produced 14 new partners for a specific VHand 13 new partners for a specific VL, which were strong binders asdetermined by ELISA.

Thus, in certain aspects, provided herein is an antibody, whichimmunospecifically binds to a D4/D5 region of a KIT polypeptide (e.g.,human KIT polypeptide), comprising a VL CDR1, VL CDR2, and VL CDR3having the amino acid sequences of SEQ ID NOs: 20, 21, and 22,respectively. In certain embodiments, provided herein is an antibody,which immunospecifically binds to a D4/D5 region of a KIT polypeptide(e.g., human KIT polypeptide), comprising a VH CDR1, VH CDR2, and VHCDR3 having the amino acid sequences of SEQ ID NOs: 23, 24, and 25,respectively. In particular embodiments, provided herein is an antibody,which immunospecifically binds to a D4/D5 region of a KIT polypeptide(e.g., human KIT polypeptide), comprising a VL domain having the aminoacid sequence of SEQ ID NO: 2, respectively. In some embodiments,provided herein is an antibody, which immunospecifically binds to aD4/D5 region of a KIT polypeptide (e.g., human KIT polypeptide),comprising a VH domain having the amino acid sequence of SEQ ID NO: 3 or5.

In certain aspects, the CDRs of an antibody described herein isdetermined according to the method of Chothia and Lesk, 1987, J. Mol.Biol., 196:901-917, which will be referred to herein as the “ChothiaCDRs” (see also, e.g., U.S. Pat. No. 7,709,226). Using the Kabatnumbering system of numbering amino acid residues in the VH chain regionand VL chain region, Chothia CDRs within an antibody heavy chainmolecule are typically present at amino acid positions 26 to 32(“CDR1”), amino acid positions 53 to 55 (“CDR2”), and amino acidpositions 96 to 101 (“CDR3”). Using the Kabat numbering system ofnumbering amino acid residues in the VH chain region and VL chainregion, Chothia CDRs within an antibody light chain molecule aretypically present at amino acid positions 26 to 33 (CDR1), amino acidpositions 50 to 52 (CDR2), and amino acid positions 91 to 96 (CDR3).

In a specific embodiment, the position of a CDR along the VH and/or VLregion of an antibody described herein may vary by one, two, three orfour amino acid positions so long as immunospecific binding to KIT(e.g., the D4/D5 region of human KIT) is maintained (e.g., substantiallymaintained, for example, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%). For example, in one embodiment,the position defining a CDR of antibody 37M or 37C may vary by shiftingthe N-terminal and/or C-terminal boundary of the CDR by one, two, three,or four, amino acids, relative to the CDR position depicted in FIGS.3A-5C, so long as immunospecific binding to KIT (e.g., the D4/D5 region)is maintained (e.g., substantially maintained, for example, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%).

TABLE 1 VL CDR Amino Acid Sequences SEQ ID Ab 37M/CVL CDR amino acid sequence NO: VL CDR1 KASQNVRTNVA 20 VL CDR2 SASYRYS 21VL CDR3 QQYNSYPRT 22

TABLE 2 VL FR Amino Acid Sequences VL Framework Region SEQ ID Ab 37M/Camino acid sequence NO: VL FR1 DIVMTQSQKFMSTSVGDRVSVTC 26 VL FR2WYQQKPGQSPKALIY 27 VL FR3 GVPDRFTGSGSGTDFTLTISNVQ 28 SEDLADYFC VL FR4FGGGTKLEIKR 29

TABLE 3 VH CDR Amino Acid Sequences SEQ ID Ab 37M/CVH CDR amino acid sequence NO: VH CDR1 DYYIN 23 VH CDR2RIYPGSGNTYYNEKFKG 24 VH CDR3 GVYYFDY 25

TABLE 4 VH FR Amino Acid Sequences SEQ ID Ab VH amino acid sequence NO:37M VH FR1 QVQLKQSGAELVRPGASVKLSCK 30 ASGYTFT VH FR2 WVKQRPGQGLEWIA 31VH FR3 KATLTAEKSSSTAYMQLSSLTSE 32 DSAVYFCAR VH FR4 WGQGTTLTVSS 33 37CVH FR1 QVQLKQSGAELVRPGASVKLSCK 38 ASGYTFT VH FR2 WVKQRPGQGLEWIA 39VH FR3 KATLTAEKSSSTAYMQLSSLTSED 40 SAVYFCAR VH FR4 WGQGTTLTVSA 41

In certain aspects, an antibody described herein comprises one or moreVL framework regions (FRs) having the amino acid sequence describedherein (e.g., see Table 2), wherein the antibody immunospecificallybinds to a KIT polypeptide, e.g., a human KIT polypeptide, for example,a D4/D5 region of KIT (e.g., human KIT), for example SEQ ID NO: 15. Inspecific embodiments, an antibody (e.g., murine, chimeric or humanizedantibody) described herein comprises a VL chain region comprising FR1,CDR1, FR2, CDR2, FR3, CDR3, and FR4. In certain embodiments, the VL FR1has the amino acid sequence of SEQ ID NO: 26. In some embodiments, theVL FR2 has the amino acid sequence of SEQ ID NO: 27. In someembodiments, the VL FR3 has the amino acid sequence of SEQ ID NO: 28. Insome embodiments, the VL FR4 has the amino acid sequence of SEQ ID NO:29.

In certain embodiments, an anti-KIT antibody described herein comprisesone or more VH FRs having the amino acid sequence described herein(e.g., see Table 4). In specific embodiments, an antibody (e.g., murine,chimeric or humanized antibody) comprises a VH chain region comprisingFR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, respectively. In someembodiments, the VH FR1 has the amino acid sequence of SEQ ID NO: 30 or38. In some embodiments, the VH FR2 has the amino acid sequence of SEQID NO: 31 or 39. In some embodiments, the VH FR3 has the amino acidsequence of SEQ ID NO: 32 or 40. In some embodiments, the VH FR4 has theamino acid sequence of SEQ ID NO: 33 or 41.

In a particular embodiment, an antibody described herein, comprises a VHFR1 having the amino acid sequence of SEQ ID NO: 38. In a particularembodiment, an antibody described herein, comprises a VH FR2 having theamino acid sequence of SEQ ID NO: 39. In a particular embodiment, anantibody described herein, comprises a VH FR3 having the amino acidsequence of SEQ ID NO: 40. In a particular embodiment, an antibodydescribed herein, comprises a VH FR4 having the amino acid sequence ofSEQ ID NO: 41.

In another specific embodiment, an antibody described herein comprises(i) a VL chain region comprising VL FR1, VL FR2, VL FR3, and VL FR4having the amino acid sequences of SEQ ID NO: 26, 27, 28, and 29,respectively; and (ii) a VH chain region comprising VH FR1, VH FR2, VHFR3, and VH FR4 having the amino acid sequences of SEQ ID NO: 30, 31,32, and 33, respectively. In a specific embodiment, an antibodydescribed herein comprises a VL chain region comprising VL FR1, VL FR2,VL FR3, and VL FR4 having the amino acid sequences of SEQ ID NO: 26, 27,28, and 29, respectively. In another specific embodiment, an antibodydescribed herein comprises a VH chain region comprising VH FR1, VH FR2,VH FR3, and VH FR4 having the amino acid sequences of SEQ ID NO: 38, 39,40, and 41, respectively.

In specific embodiments, an antibody described herein, whichimmunospecifically bind to a KIT polypeptide, e.g., a human KITpolypeptide, for example, a D4/D5 region of KIT (e.g., human KIT, forexample SEQ ID NO: 15), comprises framework regions (e.g., frameworkregions of the VL domain and/or VH domain) that are human frameworkregions or derived from human framework regions. Non-limiting examplesof human framework regions are described in the art, e.g., see Kabat etal. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242). In certain embodiment, an antibody described hereincomprises framework regions (e.g., framework regions of the VL domainand/or VH domain) that are primate (e.g., non-human primate) frameworkregions or derived from primate (e.g., non-human primate) frameworkregions.

In specific aspects, provided herein is an antibody comprising anantibody light chain and heavy chain, e.g., a separate light chain andheavy chain. With respect to the light chain, in a specific embodiment,the light chain of an antibody described herein is a kappa light chain.In another specific embodiment, the light chain of an antibody describedherein is a lambda light chain. In yet another specific embodiment, thelight chain of an antibody described herein is a human kappa light chainor a human lambda light chain. In a particular embodiment, an antibodydescribed herein, which immunospecifically binds to a KIT polypeptide(e.g., a KIT polypeptide comprising a D4/D5 region of KIT, for examplehuman KIT (e.g., SEQ ID NO: 15)) comprises a light chain wherein theamino acid sequence of the VL chain region comprises any amino acidsequence described herein (e.g., SEQ ID NO: 2), and wherein the constantregion of the light chain comprises the amino acid sequence of a humankappa light chain constant region. In another particular embodiment, anantibody described herein, which immunospecifically binds to a KITpolypeptide (e.g., a KIT polypeptide comprising a KIT polypeptidecomprising a D4/D5 region of KIT, for example human KIT (e.g., SEQ IDNO: 15)) comprises a light chain wherein the amino acid sequence of theVL chain region can comprise any amino acid sequence described herein(e.g., SEQ ID NO: 2), and wherein the constant region of the light chaincomprises the amino acid sequence of a human lambda light chain constantregion. Non-limiting examples of human constant region sequences havebeen described in the art, e.g., see U.S. Pat. No. 5,693,780 and Kabatet al. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242. In a specific embodiment, an antibody described hereincomprises a light chain comprising the amino acid sequence of SEQ ID NO:6 (or the amino acid sequence of SEQ ID NO: 6 starting at position 20,lacking the signal peptide).

In a particular embodiment, an antibody described herein, whichimmunospecifically binds to a KIT polypeptide (e.g., a KIT polypeptidecomprising a KIT polypeptide comprising a D4/D5 region of KIT, forexample human KIT (e.g., SEQ ID NO: 15)), comprises a light chaincomprising a constant region having the amino acid sequence of SEQ IDNO: 12 (TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC).

With respect to the heavy chain, in a specific embodiment, the heavychain of an antibody described herein can be an alpha (α), delta (δ),epsilon (ε), gamma (γ) or mu (μ) heavy chain. In another specificembodiment, the heavy chain of an antibody described can comprise ahuman alpha (α), delta (δ), epsilon (ε), gamma (γ) or mu (μ) heavychain. In a particular embodiment, an antibody described herein, whichimmunospecifically binds to a KIT polypeptide (e.g., a KIT polypeptidecomprising a KIT polypeptide comprising a D4/D5 region of KIT, forexample human KIT (e.g., SEQ ID NO: 15)), comprises a heavy chainwherein the amino acid sequence of the VH chain region can comprise anyamino acid sequence described herein (e.g., any of SEQ ID NOs: 3 and 5),and wherein the constant region of the heavy chain comprises the aminoacid sequence of a human gamma (γ) heavy chain constant region.Non-limiting examples of human constant region sequences have beendescribed in the art, e.g., see U.S. Pat. No. 5,693,780 and Kabat et al.(1991) Sequences of Proteins of Immunological Interest, Fifth Edition,U.S. Department of Health and Human Services, NIH Publication No.91-3242. In a specific embodiment, an antibody described hereincomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:7.

In a particular embodiment, an antibody described herein, whichimmunospecifically binds to a KIT polypeptide (e.g., a KIT polypeptidecomprising a KIT polypeptide comprising a D4/D5 region of KIT, forexample human KIT (e.g., SEQ ID NO: 15)), comprises a heavy chaincomprising a constant region having the amino acid sequence of SEQ IDNO: 13 (ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK).

In a specific embodiment, an antibody described herein, whichimmunospecifically binds to a KIT polypeptide (e.g., a D4/D5 region ofKIT, for example human KIT) comprises a VL chain region and a VH chainregion comprising any amino acid sequences described herein, and whereinthe constant regions comprise the amino acid sequences of the constantregions of an IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule, ora human IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule. Inanother specific embodiment, an antibody described herein, whichimmunospecifically binds to a KIT polypeptide (e.g., a D4/D5 region ofKIT, for example human KIT) comprises a VL chain region and a VH chainregion comprising any amino acid sequences described herein, and whereinthe constant regions comprise the amino acid sequences of the constantregions of an IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule,any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), or any subclass(e.g., IgG2a and IgG2b) of immunoglobulin molecule. In a particularembodiment, the constant regions comprise the amino acid sequences ofthe constant regions of a human IgG, IgE, IgM, IgD, IgA or IgYimmunoglobulin molecule, any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulinmolecule.

In yet another specific embodiment, an antibody described herein, whichimmunospecifically binds to a KIT polypeptide (e.g., a D4/D5 region ofKIT, for example human KIT), comprises a VL chain region and a VH chainregion comprising any amino acid sequences described herein, and whereinthe constant regions comprise the amino acid sequences of the constantregions of a human IgG1 (e.g., isotype a, z, or f) or human IgG4. In aparticular embodiment, an antibody described herein, whichimmunospecifically binds to a KIT polypeptide (e.g., a D4/D5 region ofKIT, for example human KIT) comprises a VL chain region and a VH chainregion comprising any amino acid sequences described herein, and whereinthe constant regions comprise the amino acid sequences of the constantregion of a human IgG1 (isotype f). Non-limiting examples of humanconstant regions are described in the art, e.g., see Kabat et al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242. Inanother specific embodiment, an antibody described herein, whichimmunospecifically binds to a KIT polypeptide (e.g., a D4/D5 region ofKIT, for example human KIT), comprises a VL chain region and a VH chainregion comprising any amino acid sequences described herein, and whereinthe antibody further comprises (i) a light chain constant regioncomprising the amino acid sequence of SEQ ID NO: 12, and (ii) a heavychain constant region comprising the amino acid sequence of SEQ ID NO:13.

In another particular embodiment, an antibody described herein, whichimmunospecifically binds to a KIT polypeptide (e.g., a D4/D5 region ofKIT, for example human KIT), comprises a light chain and a heavy chain,wherein (i) the light chain comprises a VL chain region comprising a VLCDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NO:20, 21, and 22, respectively; (ii) the heavy chain comprises a VH chainregion comprising a VH CDR1, VH CDR2, and VH CDR3 having the amino acidsequences of SEQ ID NO: 23, 24, and 25, respectively; (iii) the lightchain further comprises a constant light chain domain comprising theamino acid sequence of the constant domain of a human kappa light chain;and (iv) the heavy chain further comprises a constant heavy chain domaincomprising the amino acid sequence of the constant domain of a humanIgG1 (optionally IgG1 (isotype 0) heavy chain. In particularembodiments, the light chain constant region comprises the amino acidsequence of SEQ ID NO: 12; and the heavy chain constant region comprisesthe amino acid sequence of SEQ ID NO: 13.

In another particular embodiment, an antibody described herein, whichimmunospecifically binds to a KIT polypeptide (e.g., a D4/D5 region ofKIT, for example human KIT), comprises a light chain and a heavy chain,wherein (i) the light chain comprises a VL chain region comprising a VLCDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NO:20, 21, and 22, respectively; (ii) the heavy chain comprises a VH chainregion comprising a VH CDR1, VH CDR2, and VH CDR3 having the amino acidsequences of SEQ ID NO: 23, 24, and 25, respectively; (iii) the lightchain further comprises a light chain constant domain comprising theamino acid sequence of SEQ ID NO: 12; and (iv) the heavy chain furthercomprises a heavy chain constant domain comprising the amino acidsequence of SEQ ID NO: 13.

In another particular embodiment, an antibody described herein, whichimmunospecifically binds to a KIT polypeptide (e.g., a D4/D5 region ofKIT, for example human KIT), comprises a light chain and a heavy chain,wherein (i) the light chain comprises a VL chain region comprising theamino acid sequence of SEQ ID NO: 2; (ii) the heavy chain comprises a VHchain region comprising the amino acid sequence of SEQ ID NO: 3; (iii)the light chain further comprises a constant domain comprising the aminoacid sequence of the constant domain of a human kappa light chain; and(iv) the heavy chain further comprises a constant domain comprising theamino acid sequence of the constant domain of a human IgG1 (optionallyIgG1 (isotype 0) heavy chain. In particular embodiments, the light chainconstant domain comprises the amino acid sequence of SEQ ID NO: 12; andthe heavy chain constant domain comprises the amino acid sequence of SEQID NO: 13.

In another particular embodiment, an antibody described herein, whichimmunospecifically binds to a KIT polypeptide (e.g., a D4/D5 region ofKIT, for example human KIT), comprises a light chain and a heavy chain,wherein (i) the light chain comprises a VL chain region comprising theamino acid sequence of SEQ ID NO: 2; (ii) the heavy chain comprises a VHchain region comprising the amino acid sequence of SEQ ID NO: 5; (iii)the light chain further comprises a constant domain comprising the aminoacid sequence of the constant domain of a human kappa light chain; and(iv) the heavy chain further comprises a constant domain comprising theamino acid sequence of the constant domain of a human IgG1 (optionallyIgG1 (isotype 0) heavy chain. In another particular embodiment, anantibody described herein, which immunospecifically binds to a KITpolypeptide (e.g., a D4/D5 region of KIT, for example human KIT),comprises a light chain and a heavy chain, wherein (i) the light chaincomprises a VL chain region comprising the amino acid sequence of SEQ IDNO: 2; (ii) the heavy chain comprises a VH chain region comprising theamino acid sequence of SEQ ID NO: 5; (iii) the light chain furthercomprises a constant domain comprising the amino acid sequence of SEQ IDNO: 12; and (iv) the heavy chain further comprises a constant domaincomprising the amino acid sequence of SEQ ID NO: 13.

In certain embodiments, with respect to any of these antibodiesdescribed herein, the VL chain region comprises human framework regionsor is derived from human framework regions. In certain otherembodiments, the VH chain region comprises human framework regions or isderived from human framework regions. In still other embodiments, the VLchain region and VH chain region comprise human framework regions.

In certain embodiments, with respect to any of these antibodiesdescribed herein, the VL chain region comprises primate (e.g., non-humanprimate) framework regions or is derived from primate (e.g., non-humanprimate) framework regions. In certain other embodiments, the VH chainregion comprises primate (e.g., non-human primate) framework regions oris derived from primate (e.g., non-human primate) framework regions. Instill other embodiments, the VL chain region and VH chain regioncomprise primate (e.g., non-human primate) framework regions.

For example, CDRs from antigen-specific non-human antibodies, typicallyof rodent origin, are grafted onto homologous human or non-human primateacceptor frameworks. In one embodiment, the non-human primate acceptorframeworks are from Old World apes. In a specific embodiment, the OldWorld ape acceptor framework is from Pan troglodytes, Pan paniscus orGorilla gorilla. In a particular embodiment, the non-human primateacceptor frameworks are from the chimpanzee Pan troglodytes. In aparticular embodiment, the non-human primate acceptor frameworks are OldWorld monkey acceptor frameworks. In a specific embodiment, the OldWorld monkey acceptor frameworks are from the genus Macaca. In a certainembodiment, the non-human primate acceptor frameworks are is derivedfrom the cynomolgus monkey Macaca cynomolgus. Non-human primateframework sequences are described in U.S. Patent Application PublicationNo. US 2005/0208625.

In certain embodiments, an antibody described herein, whichimmunospecifically binds to a KIT polypeptide (e.g., a D4/D5 region ofKIT, for example human KIT), comprises a light chain and a heavy chain,wherein (i) the light chain comprises a VL chain region comprising humanframework regions; (ii) the heavy chain comprises a VH chain regioncomprising human framework regions; (iii) the light chain furthercomprises a light chain constant domain comprising the amino acidsequence of SEQ ID NO: 12; and (iv) the heavy chain further comprises aheavy chain constant domain comprising the amino acid sequence of SEQ IDNO: 13.

In certain embodiments, an antibody described herein, whichimmunospecifically binds to a KIT polypeptide (e.g., a D4/D5 region ofKIT, for example human KIT), comprises a light chain and a heavy chain,wherein (i) the light chain comprises a VL chain region comprising VLFR1, VL FR2, VL FR3, and VL FR4 having the amino acid sequence of SEQ IDNO: 26, 27, 28, and 29, respectively; (ii) the heavy chain comprises aVH chain region comprising VH FR1, VH FR2, VH FR3, and VH FR4 having theamino acid sequence of SEQ ID NO: 30, 31, 32, and 33, respectively;(iii) the light chain further comprises a light chain constant domaincomprising the amino acid sequence of SEQ ID NO: 12; and (iv) the heavychain further comprises a heavy chain constant domain comprising theamino acid sequence of SEQ ID NO: 13.

In certain embodiments, an antibody described herein, whichimmunospecifically binds to a KIT polypeptide (e.g., a D4/D5 region ofKIT, for example human KIT), comprises a light chain and a heavy chain,wherein (i) the light chain comprises a VL chain region comprising VLFR1, VL FR2, VL FR3, and VL FR4 having the amino acid sequence of SEQ IDNO: 30, 31, 32, and 33, respectively; (ii) the heavy chain comprises aVH chain region comprising VH FR1, VH FR2, VH FR3, and VH FR4 having theamino acid sequence of SEQ ID NO: 38, 39, 40, and 41, respectively;(iii) the light chain further comprises a light chain constant domaincomprising the amino acid sequence of SEQ ID NO: 12; and (iv) the heavychain further comprises a heavy chain constant domain comprising theamino acid sequence of SEQ ID NO: 13.

In particular embodiments, an antibody described herein, whichimmunospecifically binds to a KIT polypeptide (e.g., a D4/D5 region ofKIT, for example human KIT), comprises a light chain and a heavy chain,wherein the light chain comprises the amino acid sequence of SEQ ID NO:6 (or the amino acid sequence of SEQ ID NO: 6 starting at position 20,lacking the signal peptide), and the heavy chain comprises the aminoacid sequence of SEQ ID NO: 7 (or the amino acid sequence of SEQ ID NO:7 starting at position 20, lacking the signal peptide). In a particularembodiment, the light chain comprises a signal peptide having the aminoacid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 43). In a specificembodiment, the heavy chain comprises a signal peptide having the aminoacid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 44).

In particular embodiments, an antibody described herein, whichimmunospecifically binds to a KIT polypeptide (e.g., a D4/D5 region ofKIT, for example human KIT), comprises a light chain and a heavy chain,wherein the light chain comprises the amino acid sequence of SEQ ID NO:6 starting at amino acid residue 20 of SEQ ID NO: 6, and the heavy chaincomprises the amino acid sequence of SEQ ID NO: 7 starting at amino acidresidue 20 of SEQ ID NO: 7. In particular embodiments, an antibodydescribed herein, which immunospecifically binds to a KIT polypeptide(e.g., a D4/D5 region of KIT, for example human KIT), comprises a lightchain and a heavy chain, wherein the light chain comprises the aminoacid sequence of SEQ ID NO: 6, and the heavy chain comprises the aminoacid sequence of SEQ ID NO: 7, wherein the signal peptides of the heavychain and of the light chain have been removed (i.e., the signal peptidehas been cleaved or processed).

In certain aspects, also provided herein are antibodies, whichimmunospecifically binds to a KIT polypeptide (e.g., a D4/D5 region ofKIT, for example human KIT), comprising one or more amino acid residuesubstitutions, e.g., in the VL chain region or VH chain region, forexample, the CDRs or FRs. In specific embodiments, none of the aminoacid residue substitutions are located within the CDRs. In specificembodiments, all of the amino acid substitutions are in the FRs.

In certain embodiments, an antibody described herein comprises a VLchain region having at least 80%, at least 85%, at least 90%, at least95%, or at least 98% sequence identity to the amino acid sequence of SEQID NO: 2, wherein the antibody immunospecifically binds to a KITpolypeptide, e.g., a human KIT polypeptide, for example, a D4/D5 regionof KIT (e.g., human KIT, for example SEQ ID NO: 15). In certainembodiments, an antibody described herein comprises a VL chain regionhaving at least 80%, at least 85%, at least 90%, at least 95%, or atleast 98% sequence identity to the amino acid sequence of SEQ ID NO: 2,wherein the antibody immunospecifically binds to a KIT polypeptide,e.g., a human KIT polypeptide, for example, a D4/D5 region of KIT (e.g.,human KIT, for example SEQ ID NO: 15), and wherein the antibodycomprises CDRs (e.g., VL CDRs) that are identical to the CDRs (e.g., VLCDRs) of antibody 37M or 37C (e.g., SEQ ID NO: 20, 21, and 22). In aspecific embodiment, such antibody comprises a VL CDR1 having thesequence of SEQ ID NO: 20 that does not have an amino acid substitutionat position 7 of SEQ ID NO: 20 (for example, the R amino acid atposition 7 of SEQ ID NO: 20 is not substituted with G). In a specificembodiment, such antibody comprises a VL CDR3 having the sequence of SEQID NO: 22 that does not have an amino acid substitution at position 8 ofSEQ ID NO: 22 (for example, the R amino acid at position 8 of SEQ ID NO:22 is not substituted with L). In certain embodiments, an antibodydescribed herein comprises a light having at least 80%, at least 85%, atleast 90%, at least 95%, or at least 98% sequence identity to the aminoacid sequence of SEQ ID NO: 6 (or the amino acid sequence of SEQ ID NO:6 starting at position 20, lacking the signal peptide), wherein theantibody immunospecifically binds to a KIT polypeptide, e.g., a humanKIT polypeptide, for example, a D4/D5 region of KIT (e.g., human KIT,for example SEQ ID NO: 15).

In certain embodiments, an antibody described herein comprises a VLchain region comprising VL framework regions having at least 80%, atleast 85%, at least 90%, at least 95%, or at least 98% sequence identityto the amino acid sequence of the framework regions of SEQ ID NO: 2,wherein the antibody immunospecifically binds to a KIT polypeptide,e.g., a human KIT polypeptide, for example, a D4/D5 region of KIT (e.g.,human KIT, for example SEQ ID NO: 15). In a particular embodiment, theantibody comprises VL CDRs that are identical to the VL CDRs of antibody37M or 37C (e.g., SEQ ID NO: 20, 21, and 22). In a specific embodiment,such antibody comprises a VL CDR1 having the sequence of SEQ ID NO: 20that does not have an amino acid substitution at position 7 of SEQ IDNO: 20 (for example, the R amino acid at position 7 of SEQ ID NO: 20 isnot substituted with G). In a specific embodiment, such antibodycomprises a VL CDR3 having the sequence of SEQ ID NO: 22 that does nothave an amino acid substitution at position 8 of SEQ ID NO: 22 (forexample, the R amino acid at position 8 of SEQ ID NO: 22 is notsubstituted with L).

In certain embodiments, an antibody described herein comprises a VHchain region having at least 80%, at least 85%, at least 90%, at least95%, or at least 98% sequence identity to the amino acid sequence of SEQID NO: 3 or 5, wherein the antibody immunospecifically binds to a KITpolypeptide, e.g., a human KIT polypeptide, for example, a D4/D5 regionof KIT (e.g., human KIT, for example SEQ ID NO: 15). In certainembodiments, an antibody described herein comprises a VH chain regionhaving at least 80%, at least 85%, at least 90%, at least 95%, or atleast 98% sequence identity to the amino acid sequence of SEQ ID NO: 3,wherein the antibody immunospecifically binds to a KIT polypeptide,e.g., a human KIT polypeptide, for example, a D4/D5 region of KIT (e.g.,human KIT, for example SEQ ID NO: 15). In certain embodiments, anantibody described herein comprises a VH chain region having at least80%, at least 85%, at least 90%, at least 95%, or at least 98% sequenceidentity to the amino acid sequence of SEQ ID NO: 5, wherein theantibody immunospecifically binds to a KIT polypeptide, e.g., a humanKIT polypeptide, for example, a D4/D5 region of KIT (e.g., human KIT,for example SEQ ID NO: 15). In certain embodiments, an antibodydescribed herein comprises a heavy chain having at least 80%, at least85%, at least 90%, at least 95%, or at least 98% sequence identity tothe amino acid sequence of SEQ ID NO: 7, wherein the antibodyimmunospecifically binds to a KIT polypeptide, e.g., a human KITpolypeptide, for example, a D4/D5 region of KIT (e.g., human KIT, forexample SEQ ID NO: 15). In specific embodiments, such antibody comprisesCDRs (e.g., VH CDRs) identical to the CDRs (e.g., VH CDRs) of antibody37M or 37C (e.g., SEQ ID NOs: 20-25).

In certain embodiments, an antibody described herein comprises a VHchain region comprising VH framework regions having at least 80%, atleast 85%, at least 90%, at least 95%, or at least 98% sequence identityto the amino acid sequence of the framework regions of SEQ ID NO: 3,wherein the antibody immunospecifically binds to a KIT polypeptide,e.g., a human KIT polypeptide, for example, a D4/D5 region of KIT (e.g.,human KIT, for example SEQ ID NO: 15). In specific embodiments, suchantibody comprises CDRs (e.g., VH CDRs) identical to the CDRs (e.g., VHCDRs) of antibody 37M or 37C (e.g., SEQ ID NOs: 20-25).

In certain embodiments, an antibody described herein comprises (i) a VLchain region having at least 80%, at least 85%, at least 90%, at least95%, or at least 98% sequence identity to the amino acid sequence of SEQID NO: 2, and (ii) a VH chain region having at least 80%, at least 85%,at least 90%, at least 95%, or at least 98% sequence identity to theamino acid sequence of SEQ ID NO: 3, wherein the antibodyimmunospecifically binds to a KIT polypeptide, e.g., a human KITpolypeptide, for example, a D4/D5 region of KIT (e.g., human KIT, forexample SEQ ID NO: 15). In specific embodiments, such antibody comprisesCDRs (e.g., VL CDRs and/or VH CDRs) identical to the CDRs (e.g., VL CDRsand/or VH CDRs) of antibody 37M or 37C (e.g., SEQ ID NOs: 20-25). In aspecific embodiment, such antibody comprises a VL CDR1 having thesequence of SEQ ID NO: 20 that does not have an amino acid substitutionat position 7 of SEQ ID NO: 20 (for example, the R amino acid atposition 7 of SEQ ID NO: 20 is not substituted with G). In a specificembodiment, such antibody comprises a VL CDR3 having the sequence of SEQID NO: 22 that does not have an amino acid substitution at position 8 ofSEQ ID NO: 22 (for example, the R amino acid at position 8 of SEQ ID NO:22 is not substituted with L).

In certain embodiments, an antibody described herein comprises (i) a VLchain region having at least 80%, at least 85%, at least 90%, at least95%, or at least 98% sequence identity to the amino acid sequence of SEQID NO: 2, and (ii) a VH chain region having at least 80%, at least 85%,at least 90%, at least 95%, or at least 98% sequence identity to theamino acid sequence of SEQ ID NO: 5, wherein the antibodyimmunospecifically binds to a KIT polypeptide, e.g., a human KITpolypeptide, for example, a D4/D5 region of KIT (e.g., human KIT, forexample SEQ ID NO: 15). In specific embodiments, such antibody comprisesCDRs identical to the CDRs of antibody 37M or 37C (e.g., SEQ ID NOs:20-25). In a specific embodiment, such antibody comprises a VL CDR1having the sequence of SEQ ID NO: 20 that does not have an amino acidsubstitution at position 7 of SEQ ID NO: 20 (for example, the R aminoacid at position 7 of SEQ ID NO: 20 is not substituted with G). In aspecific embodiment, such antibody comprises a VL CDR3 having thesequence of SEQ ID NO: 22 that does not have an amino acid substitutionat position 8 of SEQ ID NO: 22 (for example, the R amino acid atposition 8 of SEQ ID NO: 22 is not substituted with L).

In certain embodiments, an antibody described herein comprises (i) alight chain comprising a constant region having at least 80%, at least85%, at least 90%, at least 95%, or at least 98% sequence identity tothe amino acid sequence of SEQ ID NO: 12, and (ii) a heavy chaincomprising a constant region having at least 80%, at least 85%, at least90%, at least 95%, or at least 98% sequence identity to the amino acidsequence of SEQ ID NO: 13, wherein the antibody immunospecifically bindsto a KIT polypeptide, e.g., a human KIT polypeptide, for example, aD4/D5 region of KIT (e.g., human KIT, for example SEQ ID NO: 15). Incertain embodiments, an antibody described herein comprises a lightchain comprising a constant region having at least 80%, at least 85%, atleast 90%, at least 95%, or at least 98% sequence identity to the aminoacid sequence of SEQ ID NO: 12. In certain embodiments, an antibodydescribed herein comprises a heavy chain comprising a constant regionhaving at least 80%, at least 85%, at least 90%, at least 95%, or atleast 98% sequence identity to the amino acid sequence of SEQ ID NO: 13.In specific embodiments, such antibody comprises CDRs identical to theCDRs of antibody 37M or 37C (e.g., SEQ ID NOs: 20-25).

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoacid or nucleic acid sequence). The amino acid residues or nucleotidesat corresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=numberof identical overlapping positions/total number of positions×100%). Inone embodiment, the two sequences are the same length. In a certainembodiment, the percent identity is determined over the entire length ofan amino acid sequence or nucleotide sequence.

The determination of percent identity between two sequences (e.g., aminoacid sequences or nucleic acid sequences) can also be accomplished usinga mathematical algorithm. A preferred, non-limiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci.U.S.A. 87:2264 2268, modified as in Karlin and Altschul, 1993, Proc.Natl. Acad. Sci. U.S.A. 90:5873 5877. Such an algorithm is incorporatedinto the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol.Biol. 215:403. BLAST nucleotide searches can be performed with theNBLAST nucleotide program parameters set, e.g., for score=100,wordlength=12 to obtain nucleotide sequences homologous to a nucleicacid molecules described herein. BLAST protein searches can be performedwith the XBLAST program parameters set, e.g., to score 50, wordlength=3to obtain amino acid sequences homologous to a protein moleculedescribed herein. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al., 1997,Nucleic Acids Res. 25:3389 3402. Alternatively, PSI BLAST can be used toperform an iterated search which detects distant relationships betweenmolecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI Blastprograms, the default parameters of the respective programs (e.g., ofXBLAST and NBLAST) can be used (see, e.g., National Center forBiotechnology Information (NCBI) on the worldwide web, ncbi.nlmnih.gov). Another preferred, non limiting example of a mathematicalalgorithm utilized for the comparison of sequences is the algorithm ofMyers and Miller, 1988, CABIOS 4:11 17. Such an algorithm isincorporated in the ALIGN program (version 2.0) which is part of the GCGsequence alignment software package. When utilizing the ALIGN programfor comparing amino acid sequences, a PAM120 weight residue table, a gaplength penalty of 12, and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically only exact matches arecounted.

In particular embodiments, the glycosylation of antibodies describedherein is modified. For example, an aglycoslated antibody can be made(i.e., the antibody lacks glycosylation) or an antibody comprising amutation or substitution at one or more glycosylation sites to eliminateglycosylation at the one or more glycosylation sites can be made.Glycosylation can be altered to, for example, increase the affinity ofthe antibody for a target antigen (e.g., human KIT, for example, a D4/D5region of human KIT). Such carbohydrate modifications can beaccomplished by, for example, altering one or more sites ofglycosylation within the antibody sequence. For example, one or moreamino acid substitutions can be made that result in elimination of oneor more variable region (e.g., VL and/or VH CDRs or VL and/or VH FRs)glycosylation sites to thereby eliminate glycosylation at that site.Such aglycosylation can increase the affinity of the antibody forantigen (e.g., human KIT, for example, a D4/D5 region of human KIT).Such an approach is described in further detail in U.S. Pat. Nos.5,714,350 and 6,350,861.

Glycosylation can occur via N-linked (or asparagine-linked)glycosylation or O-linked glycosylation. N-linked glycosylation involvescarbohydrate modification at the side-chain NH₂ group of an asparagineamino acid in a polypeptide. O-linked glycosylation involvescarbohydrate modification at the hydroxyl group on the side chain of aserine, threonine, or hydroxylysine amino acid.

In specific embodiments, an asparagine (N) residue within a VH (e.g.,SEQ ID NO: 3 or 5) or VL region (e.g., SEQ ID NO: 2) of an antibodydescribed herein is substituted with a serine (S) or another amino acid(e.g., alanine, glycine, glutamine, serine, threonine, tyrosine,cysteine). In other specific embodiments, an asparagine (N) residuewithin a VH CDR (e.g., VH CDR1, VH CDR2, and/or VH CDR3) and/or a VL CDR(e.g., VL CDR1, VL CDR2, and/or VL CDR3) of an antibody described hereinis substituted with a serine (S) or another amino acid (e.g., alanine,glycine, glutamine, serine, threonine, tyrosine, cysteine). In otherspecific embodiments, an asparagine (N) residue within a VH FR (e.g., VHFR1, VH FR2, VH FR3 and/or VH FR4) and/or a VL FR (e.g., VL FR1, VL FR2,VL FR3, and/or VL FR4) of an antibody described herein is substitutedwith a serine (S) or another amino acid (e.g., alanine, glycine,glutamine, serine, threonine, tyrosine, cysteine).

In certain embodiments, aglycosylated antibodies can be produced inbacterial cells which lack the necessary glycosylation machinery. Cellswith altered glycosylation machinery have been described in the art andcan be used as host cells in which to express recombinant antibodiesdescribed herein to thereby produce an antibody with alteredglycosylation. See, for example, Shields, R. L. et al. (2002) J. Biol.Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-1, aswell as, European Patent No: EP 1,176,195; PCT Publications WO03/035835; WO 99/54342.

In certain embodiments, one or more modifications can be made to the Fcregion of an antibody described here, generally, to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. These modifications are known in the art, and aredescribed in for example, International Patent Application PublicationNo. WO 2008/153926 A2.

In specific embodiments, an asparagine (N) residue within the constantregion of a heavy chain (e.g., SEQ ID NO: 13) and/or the constant regionof a light region (e.g., SEQ ID NO: 12) of an antibody described hereinis substituted with a serine (S) or another amino acid (e.g., alanine,glycine, glutamine, serine, threonine, tyrosine, cysteine).

In specific embodiments, an asparagine (N) residue within a heavy chain(e.g., SEQ ID NO: 7) and/or a light region (e.g., SEQ ID NO: 6) of anantibody described herein is substituted with a serine (S) or anotheramino acid (e.g., alanine, glycine, glutamine, serine, threonine,tyrosine, cysteine).

Provided herein are antibodies that immunospecifically bind to KITantigen and that can modulate KIT activity. In certain embodiments, anantibody provided herein immunospecifically binds to a KIT polypeptide,e.g., a human KIT polypeptide, and inhibits a KIT activity. KIT activitycan relate to any activity of KIT known or described in the art, e.g.,KIT receptor dimerization, KIT receptor phosphorylation (tyrosinephosphorylation), signaling downstream of the KIT receptor (e.g., AKT,MAPK/ERK, Ras, Stat1, Stat3, or Stat5 signaling), KIT ligand (e.g., SCF)induced transcriptional regulation (e.g., SCF-induced transcriptionalactivation of c-Myc), induction or enhancement of cell proliferation, orcell survival. KIT activity or KIT function are used interchangeablyherein. In certain aspects, KIT activity is induced by KIT ligand (e.g.,SCF) binding to KIT receptor. In particular aspects, KIT activity can beinduced or enhanced by gain-of-function mutations which can result, forexample, in dimerization and constitutively active KIT signaling (see,e.g., Mol et al., J. Biol. Chem., 2003, 278:31461-31464; Hirota et al.,J. Pathology, 2001, 193:505-510). Such gain-of-function can allow forKIT receptor dimerization and KIT signaling to occur in the absence ofKIT ligand (e.g., SCF) binding to KIT receptor. In certain embodiments,an increase in KIT activity or signaling can occur, in the absence ofKIT ligand (e.g., SCF) binding KIT receptor, due to high (oroverexpression) expression of KIT receptors. High or overexpression ofKIT in a cell refers to an expression level which is at least about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% more than the expressionlevel of a reference cell known to have normal KIT expression or KITactivity or more than the average expression level of KIT in apopulation of cells or samples known to have normal KIT expression orKIT activity. Expression levels of KIT can be assessed by methodsdescribed herein or known to one of skill in the art (e.g., Westernblotting or immunohistochemistry). In particular embodiments, KITactivity that is higher than normal KIT activity can lead to cellulartransformation, neoplasia, and tumorogenesis. In particular embodiments,KIT activity that is higher than normal KIT activity can lead to otherKIT-mediated disorders or diseases.

In certain embodiments, an anti-KIT antibody described herein does notblock or inhibit binding of KIT ligand (e.g., SCF) to KIT receptor. Incertain embodiments, an anti-KIT antibody described herein onlynegligibly (e.g., less than about 2% or 3%) inhibits or reduces bindingof KIT ligand (e.g., SCF) to KIT receptor. In certain embodiments, ananti-KIT antibody described herein does not induce or enhancedissociation of KIT ligand (e.g., SCF) from the KIT receptor. In certainembodiments, an anti-KIT antibody described herein only negligibly(e.g., less than about 2% or 3%) induces or enhances dissociation of KITligand (e.g., SCF) from the KIT receptor.

In specific embodiments, antibodies described herein specifically bindto an extracellular domain of KIT (e.g., D4/D5 region of KIT, forexample human KIT) and block or inhibit (e.g., partially inhibit)binding of KIT ligand (e.g., SCF) to KIT by at least about 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 98%, as assessed by methods described herein or known toone of skill in the art, e.g., ELISA assay, flow cytometry, orcompetition assay.

In certain aspects, inhibition by anti-KIT antibodies described herein(e.g., monoclonal antibody) of KIT ligand (e.g., SCF) binding to KIT canbe characterized by IC₅₀ values, which reflects the concentration ofanti-KIT antibodies achieving 50% inhibition of binding of KIT ligand toKIT. Thus, in specific embodiments, an anti-KIT antibody describedherein (e.g., antibody 37M or 37C or an antigen-binding fragmentthereof, or an antibody comprising CDRs of antibody 37M, or a conjugatecomprising, for example, antibody 37M or 37C or a KIT-binding fragmentthereof linked, covalently or noncovalently, to a therapeutic agent)inhibits binding of KIT ligand to KIT with an IC₅₀ of at most about10,000 nM, 1,000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300nM, 200 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20nM, 10 nM, 5 nM, 1 nM, 0.75 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, 0.005nM, or 0.001 nM, as assessed by methods described herein and/or known toone of skill in the art, (e.g., ELISA assay or flow cytometry). Inspecific embodiments, an anti-KIT antibody described herein inhibitsbinding of KIT ligand to KIT with an IC₅₀ of at least about 10,000 nM,1,000 nM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10nM, 5 nM, 1 nM, 0.75 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, 0.005 nM, or0.001 nM, as assessed by methods described herein and/or known to one ofskill in the art, (e.g., ELISA assay or flow cytometry). In particularembodiments, an anti-KIT antibody described herein inhibits binding ofKIT ligand to KIT with an IC₅₀ in the range of about 0.01 nM to 10,000nM, 0.01 nM to 1,000 nM, 0.1 nM to 500 nM, 0.1 nM to 100 nM, or 0.1 nMto 50 nM, as assessed by methods described herein and/or known to one ofskill in the art, (e.g., ELISA assay or flow cytometry).

In certain embodiments, an anti-KIT antibody described herein (e.g.,antibody 37M or 37C or an antigen-binding fragment thereof, or anantibody comprising CDRs of antibody 37M, or a conjugate comprising, forexample, antibody 37M or 37C or a KIT-binding fragment thereof linked,covalently or noncovalently, to a therapeutic agent) does not block orinhibit KIT receptor dimerization. In certain embodiments, an anti-KITantibody described herein only negligibly (e.g., less than about 2% or3% or within a standard of error or deviation) inhibits or reduces KITreceptor dimerization. In certain embodiments, an anti-KIT antibodydescribed herein does not induce or enhance KIT receptor dimerdissociation. In certain embodiments, an anti-KIT antibody describedherein only negligibly (e.g., less than about 2% or 3% or within astandard of error or deviation) induces or enhances KIT receptor dimerdissociation. In a particular embodiment, an anti-KIT antibody describedherein can specifically bind to a KIT receptor dimer and do not block orinhibit KIT receptor dimerization. In a particular embodiment, ananti-KIT antibody described herein can specifically bind to a KITreceptor monomer and do not block or inhibit KIT receptor dimerization.

In certain aspects, as an inhibitor of KIT activity, an antibodydescribed herein can block or inhibit (e.g., partially inhibit)dimerization of KIT. Generally, KIT receptor dimerization is inducedwhen KIT ligand binds to KIT. Thus, in specific embodiments, antibodiesdescribed herein specifically bind to KIT and block or inhibit (e.g.,partially inhibit) dimerization of KIT receptors by at least about 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 98%, or 99% as assessed by methods described hereinor known to one of skill in the art, e.g., immunoprecipitation assay,relative to dimerization of KIT receptors in the presence of KIT ligandstimulation without any antibody or with an unrelated antibody (e.g., anantibody that does not immunospecifically bind to KIT). In a specificembodiment, antibodies described herein (e.g., antibody 37M or 37C or anantigen-binding fragment thereof, or an antibody comprising CDRs ofantibody 37M, or a conjugate comprising, for example, antibody 37M or37C or a KIT-binding fragment thereof linked, covalently ornoncovalently, to a therapeutic agent) specifically bind to KIT andpartially inhibit dimerization of KIT receptors by about 25% to 75%.Blocking or inhibition (e.g., partial inhibition) of dimerization of KITreceptors by antibodies described herein can be assessed in thepresences of KIT ligand stimulation. For example, cells expressing KITare contacted with KIT ligand in the presence or absence of anti-KITantibodies described herein, and the level of KIT receptor dimerizationis determined. In certain embodiments, KIT ligand induced KIT receptordimerization in the absence of anti-KIT antibody is at least about 1fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold,3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80fold, 90 fold, or 100 fold higher than KIT receptor dimerization in thepresence of anti-KIT antibody as assessed by methods described herein orknown to one of skill in the art (e.g., immunoprecipitation assays).Tyrosine phosphorylation of one or more residues in the cytoplasmicdomain of KIT can be an indicator of KIT receptor dimerization.

In certain embodiments, an antibody described herein can inhibit (e.g.,partially inhibit) KIT activity by at least about 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98%, or 99% as assessed by methods described herein and/or known to oneof skill in the art, relative to KIT activity in the presence of KITligand stimulation without any antibody or with an unrelated antibody(e.g., an antibody that does not immunospecifically bind to KIT). Incertain embodiments, an antibody described herein can inhibit (e.g.,partially inhibit) KIT activity by at least about 25% to about 65% asassessed by methods described herein and/or known to one of skill in theart, relative to KIT activity in the presence of KIT ligand stimulationwithout any antibody or with an unrelated antibody (e.g., an antibodythat does not immunospecifically bind to KIT). Non-limiting examples ofKIT activity can include KIT receptor phosphorylation, KIT receptorsignaling, KIT ligand (e.g., SCF) mediated cell proliferation, KITligand (e.g., SCF) mediated cell survival, and transcriptionalactivation of a KIT target gene (e.g., c-Myc).

As an inhibitor of KIT activity, an antibody described herein (e.g.,antibody 37M or 37C or an antigen-binding fragment thereof, or anantibody comprising CDRs of antibody 37M, or a conjugate comprising, forexample, antibody 37M or 37C or a KIT-binding fragment thereof linked,covalently or noncovalently, to a therapeutic agent) can block (e.g.,partially block) or inhibit (e.g., partially inhibit) phosphorylation ofKIT, specifically tyrosine phosphorylation of one or more residues inthe cytoplasmic domain of KIT. Generally, KIT receptor dimerization andphosphorylation is induced when KIT ligand binds to KIT. However, incertain aspects, KIT receptor dimerization and/or phosphorylation canoccur independently of KIT ligand binding to KIT receptor. For exampleKIT receptor dimerization and/or phosphorylation can occur due togain-of-function mutations or overexpression of KIT.

Non-limiting examples of tyrosine residues in the cytoplasmic domain ofmurine KIT that can be phosphorylated, e.g., upon ligand stimulation,include 544, 546, 552, 567, 569, 577, 608, 645, 671, 674, 702, 719, 728,745, 772, 821, 844, 853, 868, 878, 898, and 934 (see Ueda et al., Blood,2002, 99:3342-3349). The corresponding tyrosine residues in thecytoplasmic domain of human KIT can be readily determined. Non-limitingexamples of tyrosine residues in the cytoplasmic domain of human KIT(e.g., GenBank Accession No. P10721) that can be phosphorylated, e.g.,upon ligand stimulation, include residues 568, 570, 703, 721, 730, 747,823, 900, and 936. In a specific embodiment, an anti-KIT antibodydescribed herein can inhibit receptor phosphorylation at tyrosineresidue 719 of murine KIT. In another specific embodiment, an anti-KITantibody described herein can inhibit receptor phosphorylation attyrosine residue 703 or 721 of human KIT.

Thus, in specific embodiments, antibodies described herein (e.g.,antibody 37M or 37C or an antigen-binding fragment thereof, or anantibody comprising CDRs of antibody 37M, or a conjugate comprising, forexample, antibody 37M or 37C or a KIT-binding fragment thereof linked,covalently or noncovalently, to a therapeutic agent) specifically bindto KIT and block or inhibit tyrosine phosphorylation in the cytoplasmicdomain of KIT by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% asassessed by methods described herein or known to one of skill in theart, e.g., ELISA assay as described in section 6 or immunoblottingassay, relative to phosphorylation in the presence of KIT ligandstimulation without any antibody or with an unrelated antibody (e.g., anantibody that does not immunospecifically bind to KIT). In particularembodiments, antibodies described herein specifically bind to KIT andblock or inhibit tyrosine phosphorylation in the cytoplasmic domain ofKIT by at least about 25%, optionally to about 65% or 75%, as assessedby methods described herein or known to one of skill in the art, e.g.,ELISA assay as described in section 6 or immunoblotting assay. Incertain embodiments, antibodies described herein specifically bind toKIT and block or inhibit tyrosine phosphorylation of the cytoplasmicdomain of KIT by at least about 25% to about 80% as assessed by methodsdescribed herein or known to one of skill in the art, e.g., ELISA assayas described in section 6 or immunoblotting assay. In specificembodiments, antibodies described herein specifically bind to KIT andblock or inhibit tyrosine phosphorylation of the cytoplasmic domain ofKIT with an IC₅₀ of less than about 400 pM or less than about 500 pM asassessed by methods described herein (e.g., phosphorylation inhibitionassay with CHO cells expressing wild-type KIT as described in Section 6below) or known to one of skill in the art. In specific embodiments,antibodies described herein specifically bind to KIT and block orinhibit tyrosine phosphorylation of the cytoplasmic domain of KIT withan IC₅₀ of less than about 200 pM. In specific embodiments, antibodiesdescribed herein specifically bind to KIT and block or inhibit tyrosinephosphorylation of the cytoplasmic domain of KIT with an IC₅₀ of lessthan about 150 pM. In specific embodiments, antibodies described hereinspecifically bind to KIT and block or inhibit tyrosine phosphorylationof the cytoplasmic domain of KIT with an IC₅₀ in the range of about 100pM to about 500 pM, about 25 pM to about 200 pM, or about 40 pM to about160 pM, or about 50 pM to about 125 pM. For example, an IC₅₀ forinhibition of tyrosine phosphorylation can be determined by assayinglysates from cells, e.g., CHO cells, recombinantly expressing KIT, inELISA which detects tyrosine phosphorylation, for example, as describedin Section 6 below. In certain embodiments, cells, e.g., CHO cells,recombinantly expressing KIT, are sorted, e.g., sorted to select forcells highly expressing KIT, prior to use in the phosphorylationinhibition assays. In some embodiments, the cells are not sorted priorto use in the phosphorylation inhibition assays.

In specific embodiments, antibodies described herein (e.g., antibody 37Mor 37C or an antigen-binding fragment thereof, or an antibody comprisingCDRs of antibody 37M, or a conjugate comprising, for example, antibody37M or 37C or a KIT-binding fragment thereof linked, covalently ornoncovalently, to a therapeutic agent) specifically bind to KIT andreduce tyrosine phosphorylation of the cytoplasmic domain of KIT by atleast about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% as assessed by methodsdescribed herein or known to one of skill in the art, e.g., ELISA assayas described in section 6 or immunoblotting assay, relative tophosphorylation in the presence of KIT ligand stimulation without anyantibody or with an unrelated antibody (e.g., an antibody that does notimmunospecifically bind to KIT). In specific embodiments, antibodiesdescribed herein (e.g., antibody 37M or 37C or an antigen-bindingfragment thereof, or an antibody comprising CDRs of antibody 37M, or aconjugate comprising, for example, antibody 37M or 37C or a KIT-bindingfragment thereof linked, covalently or noncovalently, to a therapeuticagent) specifically bind to KIT and reduce tyrosine phosphorylation ofthe cytoplasmic domain of KIT by at least about 25% or 35%, optionallyto about 75% as assessed by methods described herein or known to one ofskill in the art, e.g., ELISA assay as described in section 6 orimmunoblotting assay, relative to phosphorylation in the presence of KITligand stimulation without any antibody or with an unrelated antibody(e.g., an antibody that does not immunospecifically bind to KIT).

In specific embodiments, antibodies described herein specifically bindto KIT and block or inhibit phosphorylation of one or more tyrosineresidues in the cytoplasmic domain of KIT by at least about 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 98%, or 99% as assessed by methods described herein orknown to one of skill in the art, e.g., immunoblotting assay, relativeto phosphorylation in the presence of KIT ligand stimulation without anyantibody or with an unrelated antibody (e.g., an antibody that does notimmunospecifically bind to KIT). In specific embodiments, blocking orinhibition (e.g., partial inhibition) of phosphorylation of one or moretyrosine residues of the cytoplasmic domain of KIT by antibodiesdescribed herein can be assessed upon KIT ligand stimulation. Forexample, cells expressing KIT are contacted with KIT ligand in thepresence or absence of anti-KIT antibodies described herein, and thelevel of phosphorylation of one or more tyrosine residues in thecytoplasmic domain of KIT can be determined. In certain embodiments, KITligand induced phosphorylation of one or more tyrosine residues of thecytoplasmic domain of KIT the absence of anti-KIT antibody is at leastabout 1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold,3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70fold, 80 fold, 90 fold, or 100 fold higher than KIT ligand inducedphosphorylation of one or more tyrosine residues of the cytoplasmicdomain of KIT in the presence of anti-KIT antibody, as assessed bymethods described herein or known to one of skill in the art (e.g.,immunoblotting assays), relative to phosphorylation in the presence ofKIT ligand stimulation without any antibody or with an unrelatedantibody (e.g., an antibody that does not immunospecifically bind toKIT).

In specific embodiments, antibodies described herein (e.g., antibody 37Mor 37C or an antigen-binding fragment thereof, or an antibody comprisingCDRs of antibody 37M, or a conjugate comprising, for example, antibody37M or 37C or a KIT-binding fragment thereof linked, covalently ornoncovalently, to a therapeutic agent) specifically bind to KIT andinduce or enhance KIT receptor internalization by at least about 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 98%, or 99% as assessed by methods described hereinor known to one of skill in the art, relative to internalization in thepresence of an unrelated antibody (e.g., an antibody that does notimmunospecifically bind to KIT). In specific embodiments, antibodiesdescribed herein (e.g., antibody 37M or 37C or an antigen-bindingfragment thereof, or an antibody comprising CDRs of antibody 37M, or aconjugate comprising, for example, antibody 37M or 37C or a KIT-bindingfragment thereof linked, covalently or noncovalently, to a therapeuticagent) specifically bind to KIT and induce or enhance KIT receptorinternalization by at least about 25% or 35%, optionally to about 75%,as assessed by methods described herein or known to one of skill in theart, relative to internalization in the presence of an unrelatedantibody (e.g., an antibody that does not immunospecifically bind toKIT). In specific embodiments, antibodies described herein specificallybind to KIT and induce or enhance KIT receptor internalization by atleast about 1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60fold, 70 fold, 80 fold, 90 fold, or 100 fold as assessed by methodsdescribed herein or known to one of skill in the art, relative tointernalization in the presence of an unrelated antibody (e.g., anantibody that does not immunospecifically bind to KIT). Techniques forthe quantitation or visualization of cell surface receptors are wellknown in the art and include a variety of fluorescent and radioactivetechniques. For example, one method involves incubating the cells with aradiolabeled anti-receptor antibody. Alternatively, the natural ligandof the receptor can be conjugated to a fluorescent molecule orradioactive-label and incubated with the cells. Additional receptorinternalization assays are well known in the art and are described in,for example, Jimenez et al., Biochemical Pharmacology, 1999,57:1125-1131; Bernhagen et al., Nature Medicine, 2007, 13:587-596; andConway et al., J. Cell Physiol., 2001, 189:341-55.

In specific embodiments, antibodies described herein (e.g., antibody 37Mor 37C or an antigen-binding fragment thereof, or an antibody comprisingCDRs of antibody 37M, or a conjugate comprising, for example, antibody37M or 37C or a KIT-binding fragment thereof linked, covalently ornoncovalently, to a therapeutic agent) specifically bind to KIT andinhibit or reduce KIT receptor internalization by at least about 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 98%, or 99% as assessed by methods described hereinor known to one of skill in the art, relative to internalization in thepresence of an unrelated antibody (e.g., an antibody that does notimmunospecifically bind to KIT). In specific embodiments, antibodiesdescribed herein (e.g., antibody 37M or 37C or an antigen-bindingfragment thereof, or an antibody comprising CDRs of antibody 37M, or aconjugate comprising, for example, antibody 37M or 37C or a KIT-bindingfragment thereof linked, covalently or noncovalently, to a therapeuticagent) specifically bind to KIT and inhibit or reduce KIT receptorinternalization by at least about 25% or 35%, optionally to about 75%,as assessed by methods described herein or known to one of skill in theart, relative to internalization in the presence of an unrelatedantibody (e.g., an antibody that does not immunospecifically bind toKIT). In specific embodiments, antibodies described herein specificallybind to KIT and inhibit or reduce KIT receptor internalization by atleast about 1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60fold, 70 fold, 80 fold, 90 fold, or 100 fold as assessed by methodsdescribed herein or known to one of skill in the art, relative tointernalization in the presence of an unrelated antibody (e.g., anantibody that does not immunospecifically bind to KIT).

In specific embodiments, antibodies described herein specifically bindto KIT and induce or enhance KIT receptor turnover by at least about 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 98%, or 99% as assessed by methods described hereinor known to one of skill in the art (e.g., pulse-chase assay), relativeto turnover in the presence of an unrelated antibody (e.g., an antibodythat does not immunospecifically bind to KIT). In specific embodiments,antibodies described herein (e.g., antibody 37M or 37C or anantigen-binding fragment thereof, or an antibody comprising CDRs ofantibody 37M, or a conjugate comprising, for example, antibody 37M or37C or a KIT-binding fragment thereof linked, covalently ornoncovalently, to a therapeutic agent) specifically bind to KIT andinduce or enhance KIT receptor turnover by at least about 25% or 35%,optionally to about 75%, as assessed by methods described herein orknown to one of skill in the art (e.g., pulse-chase assay), relative toturnover in the presence of an unrelated antibody (e.g., an antibodythat does not immunospecifically bind to KIT). In specific embodiments,antibodies described herein specifically bind to KIT and induce orenhance KIT receptor turnover by at least about 1 fold, 1.2 fold, 1.3fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold,4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or100 fold as assessed by methods described herein or known to one ofskill in the art (e.g., pulse-chase assay), relative to turnover in thepresence of an unrelated antibody (e.g., an antibody that does notimmunospecifically bind to KIT). Methods for the determining receptorturnover are well known in the art. For example, cells expressing KITcan be pulse-labeled using ³⁵S-EXPRESS Protein Labeling mix (NEG772, NENLife Science Products), washed and chased with unlabeled medium for aperiod of time before protein lysates from the labeled cells areimmunoprecipitated using an anti-KIT antibody and resolved by SDS-PAGEand visualized (e.g., exposed to a PhosphoImager screen (MolecularDynamics), scanned using the Typhoon8600 scanner (Amersham), andanalyzed using ImageQuant software (Molecular Dynamics)) (see, e.g.,Chan et al., Development, 2004, 131:5551-5560).

In specific embodiments, antibodies described herein specifically bindto KIT and induce or enhance KIT receptor degradation by at least about5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 98%, or 99% as assessed by methods describedherein or known to one of skill in the art (e.g., pulse-chase assays),relative to degradation in the presence of an unrelated antibody (e.g.,an antibody that does not immunospecifically bind to KIT). In specificembodiments, antibodies described herein specifically bind to KIT andinduce or enhance KIT receptor degradation by at least about 25% or 35%,optionally to about 75%, as assessed by methods described herein orknown to one of skill in the art (e.g., pulse-chase assays), relative todegradation in the presence of an unrelated antibody (e.g., an antibodythat does not immunospecifically bind to KIT). In specific embodiments,antibodies described herein specifically bind to KIT and induce orenhance KIT receptor degradation by at least about 1 fold, 1.2 fold, 1.3fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold,4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or100 fold as assessed by methods described herein or known to one ofskill in the art (e.g., pulse-chase assays), relative to degradation inthe presence of an unrelated antibody (e.g., an antibody that does notimmunospecifically bind to KIT). Techniques for quantitating ormonitoring ubiquitination and/or degradation (e.g., kinetics or rate ofdegradation) of cell surface receptors are well known in the art andinvolve a variety of fluorescent and radioactive techniques (see, e.g.,International Patent Application Publication No. WO 2008/153926 A2). Forexample, pulse chase experiments or experiments using radiolabeledligands such as ¹²⁵I-SCF can be carried out to quantitatively measuredegradation of KIT.

Moreover, signaling events downstream of KIT receptor phosphorylationcan serve as indicators of KIT activity. For example, KIT ligand (e.g.,SCF) binding to its receptor KIT stimulates several distinct signalingpathways, including for example members of Src family kinases,phosphatidylinositol (PI) 3-kinases, and Ras mitogen-activated proteinkinase (MAPK) (see Munugalavadla et al., Mol. Cell. Biol., 2005,25:6747-6759). Phosphorylated tyrosines in the cytoplasmic domain of KITcan provide for binding sites for SH2 domain-containing proteins, whichinclude, but are not limited to, proteins of the p21Ras-mitogenactivated protein kinase (MAPK) pathway, the p85 subunit of PI 3-kinase,phospholipase C-gamma₁, the Grb2 adaptor protein, the Src family kinases(SFKs), Cbl, CRKL, p62Dok-1, SHP1, and SHP2 (see Ueda et al., Blood,2002, 99:3342-3349).

Thus, in certain aspects, anti-KIT antibodies described herein which actas inhibitors of KIT activity can inhibit signaling of a member of theSrc family kinases, PI 3-kinases, or Ras-MAPK. In particularembodiments, anti-KIT antibodies described herein which act asinhibitors of KIT activity can inhibit binding (or inhibit interaction),to the cytoplasmic domain of KIT, of one or more SH2 domain-containingproteins, such as proteins of the p21Ras-MAPK pathway, the p85 subunitof PI 3-kinase, phospholipase C-gamma1, the Grb2 adaptor protein, amember of the SFK, Cbl, CRKL, p62Dok-1, SHP1, and SHP2. In certainembodiments, anti-KIT antibodies described herein which act asinhibitors of KIT activity can inhibit activation by KIT of one or moreSH2 domain-containing proteins, such as proteins of the p21Ras-MAPKpathway, the p85 subunit of PI 3-kinase, phospholipase C-gamma1, theGrb2 adaptor protein, a member of the SFK, Cbl, CRKL, p62Dok-1, SHP1,and SHP2.

In particular embodiments, anti-KIT antibodies described herein whichact as inhibitors of KIT activity can inhibit downstream signaling suchas phosphorylation of MAPK, phosphorylation of AKT, or phosphorylationof Stat1, Stat3, or Stat5. Thus, in certain embodiments, an anti-KITantibody described herein can inhibit or reduce phosphorylation of MAPK(e.g., KIT ligand (e.g., SCF) induced phosphorylation of MAPK) by atleast about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% as assessed by methodsdescribed herein or known to one of skill in the art, e.g., Western blotor ELISA assay as described in section 6 or immunoblotting assay,relative to phosphorylation in the presence of KIT ligand stimulationwithout any antibody or with an unrelated antibody (e.g., an antibodythat does not immunospecifically bind to KIT). In certain embodiments,an anti-KIT antibody described herein can inhibit or reducephosphorylation of AKT (e.g., KIT ligand (e.g., SCF) inducedphosphorylation of AKT) by at least about 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or99% as assessed by methods described herein or known to one of skill inthe art, e.g., Western blot or ELISA assay as described in section 6 orimmunoblotting assay, relative to phosphorylation in the presence of KITligand stimulation without any antibody or with an unrelated antibody(e.g., an antibody that does not immunospecifically bind to KIT). Inparticular embodiments, an anti-KIT antibody described herein caninhibit or reduce phosphorylation of Stat3 (e.g., KIT ligand (e.g., SCF)induced phosphorylation of Stat3) by at least about 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 98%, or 99% as assessed by methods described herein or known to oneof skill in the art, e.g., Western blot or ELISA assay as described insection 6 or immunoblotting assay, relative to phosphorylation in thepresence of KIT ligand stimulation without any antibody or with anunrelated antibody (e.g., an antibody that does not immunospecificallybind to KIT). In particular embodiments, an anti-KIT antibody describedherein can inhibit or reduce phosphorylation of Stat1 or Stat5 (e.g.,KIT ligand (e.g., SCF) induced phosphorylation) by at least about 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 98%, or 99% as assessed by methods described hereinor known to one of skill in the art, e.g., Western blot or ELISA assayas described in section 6 or immunoblotting assay, relative tophosphorylation in the presence of KIT ligand stimulation without anyantibody or with an unrelated antibody (e.g., an antibody that does notimmunospecifically bind to KIT).

In certain aspects, an anti-KIT antibody described herein which can actas an inhibitor of KIT activity or activity can inhibit cellularproliferation of cells (e.g., TF-1 cells) that express KIT and thatrespond to KIT signaling (e.g., cells that proliferate in response toKIT ligand stimulation and KIT signaling). Cell proliferation assays aredescribed in the art and can be readily carried out by one of skill inthe art. For example, cell proliferation can be assayed by measuringBromodeoxyuridine (BrdU) incorporation (see, e.g., Hoshino et al., 1986,Int. J. Cancer 38, 369; Campana et al., 1988, J. Immunol. Meth. 107:79)or (3H) thymidine incorporation (see, e.g., Blechman et al., Cell, 1995,80:103-113; Chen, J., 1996, Oncogene 13:1395-403; Jeoung, J., 1995, J.Biol. Chem. 270:18367 73), by direct cell count at various timeintervals (e.g., 12-hour or 24-hour intervals), or by detecting changesin transcription, translation or activity of known genes such asproto-oncogenes (e.g., fos, myc) or cell cycle markers (Rb, cdc2, cyclinA, D1, D2, D3, E, etc). The levels of such protein and mRNA and activitycan be determined by any method well known in the art. For example,protein can be quantitated by known immunodiagnostic methods such asELISA, Western blotting or immunoprecipitation using antibodies,including commercially available antibodies. mRNA can be quantitatedusing methods that are well known and routine in the art, for example,using northern analysis, RNase protection, or polymerase chain reactionin connection with reverse transcription.

In specific embodiments, antibodies described herein specifically bindto KIT and inhibit cell proliferation by at least about 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 98%, or 99% as assessed by methods described herein or knownto one of skill in the art (e.g., BrdU incorporation assay). In specificembodiments, antibodies described herein specifically bind to KIT andinhibit cell proliferation by at least about 1 fold, 1.2 fold, 1.3 fold,1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold,30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100fold as assessed by methods described herein or known to one of skill inthe art (e.g., BrdU incorporation assay).

In certain aspects, an anti-KIT antibody described herein which can actas an inhibitor of KIT activity can reduce or inhibit survival of cellsthat express KIT and that respond to KIT signaling (e.g., cells thatproliferate in response to KIT ligand stimulation and KIT signaling).Cell survival assays are described in the art and can be readily carriedout by one of skill in the art. For example, cell viability can beassessed by using trypan-blue staining or other cell death or viabilitymarkers known in the art. In a specific embodiment, the level ofcellular ATP is measured to determined cell viability. In specificembodiments, cell viability is measured in three-day and seven-dayperiods using an assay standard in the art, such as the CellTiter-GloAssay Kit (Promega) which measures levels of intracellular ATP. Areduction in cellular ATP is indicative of a cytotoxic effect. Inanother specific embodiment, cell viability can be measured in theneutral red uptake assay. In other embodiments, visual observation formorphological changes can include enlargement, granularity, cells withragged edges, a filmy appearance, rounding, detachment from the surfaceof the well, or other changes. These changes are given a designation ofT (100% toxic), PVH (partially toxic—very heavy—80%), PH (partiallytoxic—heavy—60%), P (partially toxic—40%), Ps (partiallytoxic—slight—20%), or 0 (no toxicity—0%), conforming to the degree ofcytotoxicity seen. A 50% cell inhibitory (cytotoxic) concentration(IC₅₀) is determined by regression analysis of these data.

In specific embodiments, antibodies described herein specifically bindto KIT and inhibit (e.g, partially inhibit) cell survival by at leastabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% as assessed by methodsdescribed herein or known to one of skill in the art (e.g., trypan blueexclusion assay). In specific embodiments, antibodies described hereinspecifically bind to KIT and inhibit cell survival by at least about 1fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold,3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80fold, 90 fold, or 100 fold as assessed by methods described herein orknown to one of skill in the art (e.g., trypan blue assay).

In certain aspects, an anti-KIT antibody described herein, which can actas an inhibitor of KIT activity, is capable of inducing apoptosis (i.e.,programmed cell death) of cells (e.g., MO7E cells) that express KIT andthat respond to KIT signaling (e.g., cells that proliferate in responseto KIT ligand stimulation and KIT signaling). Apoptosis are described inthe art and can be readily carried out by one of skill in the art. Forexample, flow cytometry can be used to detect activated caspase 3, anapoptosis-mediating enzyme, in cells undergoing apoptosis, or Westernblotting can be used to detect cleavage of poly(ADP-ribose) polymerase(PARP) (see, e.g., Smolich et al., Blood, 2001, 97:1413-1421). Cleavageof PARP is an indicator of apoptosis. In specific embodiments,antibodies described herein specifically bind to KIT and induce orenhance apoptosis by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%as assessed by methods described herein or known to one of skill in theart (e.g., flow cytometry to detect activated caspase 3). In specificembodiments, antibodies described herein specifically bind to KIT andinduce or enhance apoptosis by at least about 1 fold, 1.2 fold, 1.3fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold,4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or100 fold as assessed by methods described herein or known to one ofskill in the art (e.g., flow cytometry to detect activated caspase 3).

In certain aspects, an anti-KIT antibody described herein, which can actas an inhibitor of KIT activity, is capable of inhibiting or decreasinganchorage independent cell growth (e.g., colony formation) by cells(e.g., H526 cells or CHO cells expressing exogenous KIT) that expressKIT and that respond to KIT signaling (e.g., cells that proliferate inresponse to KIT ligand stimulation and KIT signaling), as measured bymethods commonly known in the art, e.g., soft agar assay. In specificembodiments, antibodies described herein (e.g., antibody 37M or 37C oran antigen-binding fragment thereof, or an antibody comprising CDRs ofantibody 37M, or a conjugate comprising, for example, antibody 37M or37C or a KIT-binding fragment thereof linked, covalently ornoncovalently, to a therapeutic agent) specifically bind to KIT andinhibit or decrease anchorage independent cell growth by at least about5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 98%, or 99% as assessed by methods describedherein or known to one of skill in the art (e.g., soft agar assay). Inspecific embodiments, antibodies described herein (e.g., antibody 37M or37C or an antigen-binding fragment thereof, or an antibody comprisingCDRs of antibody 37M, or a conjugate comprising, for example, antibody37M or 37C or a KIT-binding fragment thereof linked, covalently ornoncovalently, to a therapeutic agent) specifically bind to KIT andinhibit or decrease anchorage independent cell growth by at least about25% or 35%, optionally to about 75%, as assessed by methods describedherein or known to one of skill in the art (e.g., soft agar assay). Inspecific embodiments, antibodies described herein specifically bind toKIT and inhibit or decrease anchorage independent cell growth by atleast about 1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60fold, 70 fold, 80 fold, 90 fold, or 100 fold as assessed by methodsdescribed herein or known to one of skill in the art (e.g., soft agarassay).

Cells and cell lines which are appropriate for use in the assaysdescribed herein relating to KIT activity are readily available (e.g.,ATCC) or can be readily identified using methods known in the art. Forexample, cells and/or cell lines that express KIT endogenously or thatpossess KIT signaling or activity are known to one of skill in the art.In certain embodiments, cells or cell lines that are appropriate for usein the assays described herein can express KIT, either endogenously orrecombinantly. In particular embodiments, cells or cell lines for use incell proliferation assays can express KIT, endogenously orrecombinantly, and proliferate or increase proliferation in response toKIT ligand (e.g., SCF) stimulation. Cells or cell lines for use in cellviability assays can express KIT, endogenously or recombinantly, andexert changes in cell viability in response to KIT ligand (e.g., SCF)stimulation. Cells or cell lines for use in apoptosis assays can expressKIT, endogenously or recombinantly, and exert changes in apoptosis inresponse to KIT ligand (e.g., SCF) stimulation.

Non-limiting examples of cells that can be used in the methods andassays described herein include primary cells, transformed cells, stemcells, mast cells, primordial germ cells, oocytes, spermatocytes,embryonic stem cells, hematopoietic cells, erythroleukemia cells (e.g.,F36P and TF-1 cell lines), human myeloid leukemia cell lines, such asMOTE cells; gastrointestinal stromal tumor cell lines such as ST-882,GIST430, and GIST882; neuroblastoma cell lines such as SK-N-SH, SK-SY5Y,H-EP1, SK-N-BE(2), SK-N-BE(ZkM17), SK-N-BE(2)C, LA-N-1, or LA-N-1-5s;and small cell lung carcinoma cell lines such as H526, ECC12, TMK1,MKN7, GCIY, and HGC27.

Alternatively, cells and cell lines that express KIT, e.g., human KIT,can routinely be generated recombinantly. Non-limiting examples of cellsthat can be engineered to express KIT recombinantly include COS cells,HEK 293 cells, CHO cells, fibroblasts (e.g., human fibroblasts) such asNIH3T3 cells, and MEFS. In a specific embodiment, cells for use in themethods described herein are CHO cells exogenously expressingfull-length human KIT (e.g., SEQ ID NO: 1).

In certain aspects, an anti-KIT antibody described herein, which can actas an inhibitor of KIT activity, is capable of inhibiting tumor growthor inducing tumor regression in mouse model studies. For example, tumorcell lines can be introduced into nude mice, and the mice can beadministered with anti-KIT antibodies described herein one or moretimes, and tumor progression of the injected tumor cells can bemonitored over a period of weeks and/or months. In some cases,administration of anti-KIT antibodies to the nude mice can occur priorto introduction of the tumor cell lines. Any appropriate tumor cell line(e.g., tumor cell line expressing KIT) can be used in the mousexenograft models described herein. Non-limiting examples of tumor celllines for use in these xenograft mouse models include megakaryoblasticleukemia cell lines such as MO7e; gastrointestinal stromal tumor celllines such as ST-882, GIST430, GIST48, GIST48B and GIST882; humanerythroleukemic cell lines such as HEL and TF-1; human promyelocyticleukemia cell line, HL60; neuroblastoma cell lines such as SK-N-SH,SK-SY5Y, H-EP1, SK-N-BE(2), SK-N-BE(ZkM17), SK-N-BE(2)C, LA-N-1, orLA-N-1-5s; and small cell lung carcinoma cell lines such as H526,DMS153, DMS79, ECC12, TMK1, MKN7, GCIY, and HGC27. In a specificembodiments, a tumor cell line for use in a xenograft mouse model is theGIST882, GIST430, GIST48, GIST48B, HEL, HL60, H526, DMS153, or DMS79cell line. In certain embodiments, suitable cell lines for use inxenograft tumor models can be generated by recombinantly expressing KITin cell. In specific embodiments, antibodies described herein (e.g.,antibody 37M or 37C or an antigen-binding fragment thereof, or anantibody comprising CDRs of antibody 37M, or a conjugate comprising, forexample, antibody 37M or 37C or a KIT-binding fragment thereof linked,covalently or noncovalently, to a therapeutic agent) specifically bindto KIT and inhibit tumor grow or induce tumor regression in a mousemodel by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% as assessed bymethods described herein or known to one of skill in the art. Inspecific embodiments, antibodies described herein (e.g., antibody 37M or37C or an antigen-binding fragment thereof, or an antibody comprisingCDRs of antibody 37M, or a conjugate comprising, for example, antibody37M or 37C or a KIT-binding fragment thereof linked, covalently ornoncovalently, to a therapeutic agent) specifically bind to KIT andinhibit tumor grow or induce tumor regression in a mouse model by atleast about 25% or 35%, optionally to about 75%, as assessed by methodsdescribed herein or known to one of skill in the art. In specificembodiments, antibodies described herein specifically bind to KIT andinhibit tumor grow or induce tumor regression in a mouse model by atleast about 1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60fold, 70 fold, 80 fold, 90 fold, or 100 fold as assessed by methodsdescribed herein or known to one of skill in the art. Determining tumorgrowth inhibition or tumor regression can be assessed by monitoringtumor size over a period of time, such as by physical measurement ofpalpable tumors, or other visual detection methods. For example, tumorcell lines can be generated to recombinantly express a visualizationagent, such as green fluorescent protein (GFP) or luciferase, then invivo visualization of GFP can be carried out by microscopy, and in vivovisualization of luciferase can be carried out by administeringluciferase substrate to the xenograft mice and detecting luminescent dueto the luciferase enzyme processing the luciferase substrate. The degreeor level of detection of GFP or luciferase correlates to the size of thetumor in the xenograft mice.

In certain aspects, anti-KIT antibodies described herein bindspecifically to KIT antigen and can increase survival of animals intumor xenograft models. In specific embodiments, antibodies describedherein (e.g., antibody 37M or 37C or an antigen-binding fragmentthereof, or an antibody comprising CDRs of antibody 37M, or a conjugatecomprising, for example, antibody 37M or 37C or a KIT-binding fragmentthereof linked, covalently or noncovalently, to a therapeutic agent)specifically bind to KIT and increase survival of mice in tumorxenograft models by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%as assessed by methods described herein or known to one of skill in theart. In specific embodiments, antibodies described herein (e.g.,antibody 37M or 37C or an antigen-binding fragment thereof, or anantibody comprising CDRs of antibody 37M, or a conjugate comprising, forexample, antibody 37M or 37C or a KIT-binding fragment thereof linked,covalently or noncovalently, to a therapeutic agent) specifically bindto KIT and increase survival of mice in tumor xenograft models by atleast about 25% or 35%, optionally to about 75%, as assessed by methodsdescribed herein or known to one of skill in the art. In specificembodiments, antibodies described herein specifically bind to KIT andincrease survival of mice in tumor xenograft models by at least about 1fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold,3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80fold, 90 fold, or 100 fold as assessed by methods described herein orknown to one of skill in the art. Survival can be determined by plottinga survival curve of number of surviving mice against time (e.g., days orweeks) after tumor cell line injection.

Provided herein are antibodies that immunospecifically bind a KITpolypeptide, e.g., a human KIT polypeptide, e.g., a D4/D5 region of KIT,for example, human KIT, with a particular affinity.

“Affinity” of an antibody described herein for an epitope (e.g., KITepitope) is a term well understood in the art and refers to the extent,or strength, of binding of an antibody to an epitope. Affinity can bemeasured and/or expressed in a number of ways known in the art,including, but not limited to, equilibrium dissociation constant (K_(D)or K_(d)), apparent equilibrium dissociation constant (K_(D)′ orK_(d)′), and IC₅₀ (amount needed to effect 50% inhibition in acompetition assay). It is understood that, for purposes describedherein, an affinity is an average affinity for a given population ofantibodies which bind to an epitope. Values of K_(D)′ described hereinin terms of milligram (mg) Ig per mL or mg/mL indicate mg Ig per mL ofserum, although plasma can be used. When antibody affinity is used as abasis for administration of the treatment methods described herein, orselection for the treatment methods described herein, antibody affinitycan be measured before and/or during treatment, and the values obtainedcan be used by a clinician in assessing whether a human patient is anappropriate candidate for treatment.

In specific aspects, provided herein are antibodies (e.g., antibodiescomprising the CDRs of antibody 37M or 37C or an antibody which binds tothe same epitope as that of antibody 37M or 37C) that have a highbinding affinity (e.g., antibodies having a K_(D) of less than 100 nM,50 nM, 10 nM, 1 nM, 500 pM, 200 pM, 100 pM, or 50 pM) for a KIT antigen,preferably a human KIT antigen, in particular the D4/D5 region of ahuman KIT. In a specific embodiment, an antibody described herein has anassociation rate constant or k_(on) rate (antibody (Ab)+antigen (Ag)^(k)^(on) ′→Ab-Ag) of at least 2×10⁵M⁻¹s⁻¹, at least 5×10⁵M⁻¹s⁻¹, at least10⁶M⁻¹s⁻¹, at least 5×10⁶M⁻¹s⁻¹, at least 10⁷M⁻¹s⁻¹, at least5×10⁷M⁻¹s⁻¹, or at least 10⁸M⁻¹s⁻¹. In a certain embodiment, an antibodydescribed herein has a k_(on), of at least 2×10⁵ M⁻¹s⁻¹, at least 5×10⁵M⁻¹s⁻¹, at least 10⁶M⁻¹s⁻¹, at least 5×10⁶ M⁻¹s⁻¹, at least 10⁷M⁻¹s⁻¹,at least 5×10⁷M⁻¹s⁻¹, or at least 10⁸ M⁻¹s⁻¹.

In another embodiment, an antibody described herein has a k_(off) rate((Ab-Ag)^(k) ^(off) →antibody (Ab)+antigen) of less than 10⁻¹ s⁻¹, lessthan 5×10⁻¹ s⁻¹, less than 10⁻² s⁻¹, less than 5×10⁻²s⁻¹, less than 10⁻³s⁻¹, less than 5×10⁻³ s⁻¹, less than 10⁻⁴ s⁻¹, less than 5×10⁻⁴ s⁻¹,less than 10⁻⁵ s⁻¹, less than 5×10⁻⁵ s⁻¹, less than 10⁻⁶ s⁻¹, less than5×10⁻⁶ s⁻¹, less than 10⁻⁷ s⁻¹, less than 5×10⁻⁷ s⁻¹, less than 10⁻⁸s⁻¹, less than 5×10⁻⁸ s⁻¹, less than 10⁻⁹ s⁻¹, less than 5×10⁻⁹ s⁻¹, orless than 10⁻¹⁰ s⁻¹. In a specific embodiment, an antibody describedherein has a k_(on) of less than 5×10⁻⁴ s⁻¹, less than 10⁻⁵ s⁻¹, lessthan 5×10⁻⁵ s⁻¹, less than 10⁻⁶ s⁻¹, less than 5×10⁻⁶ s⁻¹, less than10⁻⁷ s⁻¹, less than 5×10⁻⁷ s⁻¹, less than 10⁻⁸ s⁻¹, less than 5×10⁻⁸s⁻¹, less than 10⁻⁹ s⁻¹, less than 5×10⁻⁹ s⁻¹, or less than 10⁻¹⁰ s⁻¹.

In another embodiment, an antibody described herein has an affinityconstant or K_(a) (k_(on)/k_(off)) of at least 10² M⁻¹, at least 5×10²M⁻¹, at least 10³ M⁻¹, at least 5×10³ M⁻¹, at least 10⁴ M⁻¹, at least5×10⁴ M⁻¹, at least 10⁵ M⁻¹, at least 5×10⁵ M⁻¹, at least 10⁶ M⁻¹, atleast 5×10⁶ M⁻¹, at least 10⁷ M⁻¹, at least 5×10⁷M⁻¹, at least 10⁸ M⁻¹,at least 5×10⁸ M⁻¹, at least 10⁹ M⁻¹, at least 5×10⁹ M⁻¹, at least 10¹⁰M⁻¹, at least 5×10¹⁰ M⁻¹, at least 10¹¹ M⁻¹, at least 5×10¹¹ M⁻¹, atleast 10¹² M⁻¹, at least 5×10¹² M⁻¹, at least 10¹³ M⁻¹, at least 5×10¹³M⁻¹, at least 10¹⁴ M⁻¹, at least 5×10¹⁴ M⁻¹, at least 10¹⁵ M⁻¹, or atleast 5×10¹⁵ M⁻¹.

In a particular embodiment, an antibody described herein has adissociation constant or K_(D) (k_(off)/k_(on)) of less than 10⁻² M,less than 5×10⁻² M, less than 10⁻³ M, less than 5×10⁻³ M, less than 10⁻⁴M, less than 5×10⁻⁴ M, less than 10⁻⁵ M, less than 5×10⁻⁵ M, less than10⁻⁶ M, less than 5×10⁻⁶ M, less than 10⁻⁷ M, less than 5×10⁻⁷M, lessthan 10⁻⁸ M, less than 5×10⁻⁸ M, less than 10⁻⁹ M, less than 5×10⁻¹⁰ M,less than 10⁻¹⁰ M, less than 5×10⁻¹⁰ M, less than 10⁻¹¹ M, less than5×10⁻¹¹ M, less than 10⁻¹² M, less than 5×10⁻¹² M, less than 10⁻¹³ M,less than 5×10⁻¹³ M, less than 10⁻¹⁴ M, less than 5×10⁻¹⁴ M, less than10⁻¹⁵ M, or less than 5×10⁻¹⁵ M.

In specific embodiments, an antibody (e.g., antibody comprising the CDRsof antibody 37M or 37C or an antibody which binds to the same epitope asthat of antibody 37M or 37C) immunospecifically binds to a KIT antigen(e.g., a D4/D5 region of KIT, for example human KIT), and has adissociation constant (K_(D)) of less than 500,000 pM (500 nM), lessthan 100,000 pM (100 nM), less than 50,000 pM (50 nM), less than 10,000pM (10 nM), less than 3,000 pM (3 nM), less than 2,500 pM (2.5 nM), lessthan 2,000 pM, less than 1,500 pM, less than 1,000 pM, less than 750 pM,less than 500 pM, less than 250 pM, less than 200 pM, less than 150 pM,less than 100 pM, less than 75 pM as assessed using an assay describedherein or known to one of skill in the art (e.g., a Biacore™ assay)(Biacore™ International AB, Uppsala, Sweden). In a specific embodiment,an antibody described herein (e.g., antibody comprising the CDRs ofantibody 37M or 37C or an antibody which binds to the same epitope asthat of antibody 37M or 37C) immunospecifically binds to a KIT antigen(e.g., a D4/D5 region of KIT, for example human KIT), and has a K_(D) inthe range of from 25 to 100,000 pM, 25 to 75,000 pM, 25 to 50,000 pM, 25to 40,000 pM, 25 to 30,000 pM, 25 to 20,000 pM, 25 to 10,000 pM, 25 to1,000 pM, 25 to 500 pM, 25 to 250 pM, 25 to 100 pM, or 25 to 50 pM asassessed using methods described herein or known to one of skill in theart (e.g., a Biacore™ assay, assay using KinExA 3000 instrument). In aparticular embodiment, an antibody described herein (e.g., antibodycomprising the CDRs of antibody 37M or 37C or an antibody which binds tothe same epitope as that of antibody 37M or 37C) immunospecifically bindto a KIT antigen (e.g., a D4/D5 region of KIT, for example human KIT),and has a K_(D) of about 1 nM to about 25 nM, or any value in between,as assessed using methods described herein or known to one of skill inthe art (e.g., a Biacore™ assay, assay using KinExA 3000 instrument). Ina particular embodiment, antibodies described herein (e.g., antibodycomprising the CDRs of antibody 37M or 37C or an antibody which binds tothe same epitope as that of antibody 37M or 37C) immunospecifically bindto a KIT antigen (e.g., a D4/D5 region of KIT, for example human KIT),and have a K_(D) of about 100 pM to about 25 nM, or any value inbetween, as assessed using methods described herein or known to one ofskill in the art (e.g., a Biacore™ assay, assay using KinExA 3000instrument). In a particular embodiment, an antibody described herein(e.g., antibody comprising the CDRs of antibody 37M or 37C or anantibody which binds to the same epitope as that of antibody 37M or 37C)immunospecifically binds to a KIT antigen (e.g., a D4/D5 region of KIT,for example human KIT), and has a K_(D) of about 1 pM to about 250 nM,or any value in between, as assessed using methods described herein orknown to one of skill in the art (e.g., a Biacore™ assay, assay usingKinExA 3000 instrument). In a particular embodiment, an antibodydescribed herein (e.g., antibody comprising the CDRs of antibody 37M or37C or an antibody which binds to the same epitope as that of antibody37M or 37C) immunospecifically binds to KIT antigen, (e.g., a D4/D5region of KIT, for example human KIT), and has a K_(D) of about 1 nM,1.5 nM, 2 nM, 2.5 nM, 3 nM, 3.5 nM, 4 nM, 4.5 nM, 5 nM, 5.5 nM, 6 nM,6.5 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16nM, 17 nM, 18 nM, 19 nM, 20 nM, or 21 nM, as assessed using methodsdescribed herein or known to one of skill in the art (e.g., a Biacore™assay, assay using KinExA 3000 instrument). In a particular embodiment,an antibody described herein (e.g., antibody comprising the CDRs ofantibody 37M or 37C or an antibody which binds to the same epitope asthat of antibody 37M or 37C) immunospecifically binds to KIT antigen(e.g., a D4/D5 region of KIT, for example human KIT), and has a K_(D)from about 100 pM to about 10 nM, as assessed using methods describedherein or known to one of skill in the art (e.g., ELISA, assay usingKinExA 3000 instrument, or Biacore™ assay). In a particular embodiment,an antibody described herein (e.g., antibody comprising the CDRs ofantibody 37M or 37C or an antibody which binds to the same epitope asthat of antibody 37M or 37C) immunospecifically binds to KIT antigen(e.g., a D4/D5 region of KIT, for example human KIT), and has a K_(D)from about 50 pM to about 1 nM, as assessed using methods describedherein or known to one of skill in the art (e.g., ELISA, assay usingKinExA 3000 instrument, or Biacore™ assay).

In specific embodiments, an anti-KIT antibody (e.g., antibody comprisingthe CDRs of antibody 37M or 37C or an antibody which binds to the sameepitope as that of antibody 37M or 37C) immunospecifically binds to aKIT antigen (e.g., a D4/D5 region of KIT, for example human KIT), andhas a concentration at 50% binding to antigen of less than 3000 pM (3nM), less than 2500 pM (2.5 nM), less than 2000 pM, less than 1500 pM,less than 1000 pM, less than 750 pM, less than 500 pM, less than 250 pM,less than 200 pM, less than 150 pM, less than 100 pM, less than 75 pM asassessed using an assay described herein or known to one of skill in theart (e.g., solid phase ELISA as described in section 6). In a specificembodiment, an antibody described herein (e.g., antibody comprising theCDRs of antibody 37M or 37C or an antibody which binds to the sameepitope as that of antibody 37M or 37C) immunospecifically binds to aKIT antigen (e.g., a D4/D5 region of KIT, for example human KIT), andhas a concentration at 50% binding to antigen in the range of from 25 to500,000 pM (500 nM), 25 to 250,000 pM (250 nM), 25 to 100,000 pM (100nM), 25 to 75,000 pM (75 nM), 25 to 50,000 pM (50 nM), 25 to 40,000 pM(40 nM), 25 to 30,000 pM (30 nM), 25 to 20,000 pM (20 nM), 25 to 10,000pM (10 nM), 25 to 1,000 pM (1 nM), 25 to 500 pM, 25 to 250 pM, 25 to 100pM, or 25 to 50 pM as assessed using methods described herein or knownto one of skill in the art (e.g., solid phase ELISA as described insection 6). In a particular embodiment, an antibody described herein(e.g., antibody comprising the CDRs of antibody 37M or 37C or anantibody which binds to the same epitope as that of antibody 37M or 37C)immunospecifically binds to a KIT antigen (e.g., a D4/D5 region of KIT,for example human KIT), and has a concentration at 50% binding toantigen of about 1 nM to about 25 nM, or any value in between, asassessed using methods described herein or known to one of skill in theart (e.g., solid phase ELISA as described in section 6). In a particularembodiment, an antibody described herein (e.g., antibody comprising theCDRs of antibody 37M or 37C or an antibody which binds to the sameepitope as that of antibody 37M or 37C) immunospecifically binds to aKIT antigen (e.g., a D4/D5 region of KIT, for example human KIT), andhas a concentration at 50% binding to antigen of about 50 pM to about500 pM, or any value in between, as assessed using methods describedherein or known to one of skill in the art (e.g., solid phase ELISA asdescribed in section 6). In a particular embodiment, an antibodydescribed herein (e.g., antibody comprising the CDRs of antibody 37M or37C or an antibody which binds to the same epitope as that of antibody37M or 37C) immunospecifically binds to KIT antigen (e.g., a D4/D5region of KIT, for example human KIT), and has a concentration at 50%binding of about 0.5 nM, 0.25 nM, 0.1 nM, 1 nM, 1.5 nM, 2 nM, 2.5 nM, 3nM, 3.5 nM, 4 nM, 4.5 nM, 5 nM, 5.5 nM, 6 nM, 6.5 nM, 7 nM, 8 nM, 9 nM,10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 150 nM, 200nM, 250 nM, 300 nM, 350 nM, 400 nM, or 500 nM, or less, as assessedusing methods described herein or known to one of skill in the art(e.g., solid phase ELISA as described in section 6). In a particularembodiment, antibodies described herein (e.g., antibody comprising theCDRs of antibody 37M or 37C or an antibody which binds to the sameepitope as that of antibody 37M or 37C) immunospecifically bind to KITantigen (e.g., a D4/D5 region of KIT, for example human KIT), and have aconcentration at 50% binding from about 100 pM to about 10 nM, asassessed using methods described herein or known to one of skill in theart (e.g., ELISA, assay using KinExA 3000 instrument, or Biacore™assay).

In specific embodiments, an anti-KIT antibody which is anantigen-binding fragment of a whole or entire antibody, e.g., Fabfragment, has comparable affinity to KIT relative to the affinity of thewhole or entire anti-KIT antibody. In certain embodiments, an anti-KITantibody which is an antigen-binding fragment of a whole or entireantibody, e.g., Fab fragment, specifically binds to KIT and has a K_(D)that is comparable to the K_(D) of the whole or entire anti-KIT antibodyas assessed using methods described herein or known to one of skill inthe art (e.g., a Biacore™ assay, assay using KinExA 3000 instrument). Incertain embodiments, an anti-KIT antibody which is an antigen-bindingfragment of a whole or entire antibody, e.g., Fab fragment, specificallybinds to KIT and has a K_(D) that is less than the K_(D) of the whole orentire anti-KIT antibody as assessed using methods described herein orknown to one of skill in the art (e.g., a Biacore™ assay, assay usingKinExA 3000 instrument). In particular embodiments, an anti-KIT antibodywhich is an antigen-binding fragment of a whole or entire antibody,e.g., Fab fragment, specifically binds to KIT and has a K_(D) that isabout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 75%, or 85%, less thanthe K_(D) of the whole or entire anti-KIT antibody as assessed usingmethods described herein or known to one of skill in the art (e.g., aBiacore™ assay, assay using KinExA 3000 instrument). In particularembodiments, an anti-KIT antibody which is an antigen-binding fragmentof a whole or entire antibody, e.g., Fab fragment, specifically binds toKIT and has a K_(D) that is at most about 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 65% or 75% less than the K_(D) of the whole or entire anti-KITantibody as assessed using methods described herein or known to one ofskill in the art (e.g., a Biacore™ assay, assay using KinExA 3000instrument).

In certain embodiments, an anti-KIT antibody which is an antigen-bindingfragment of the native (or entire) antibody, e.g., Fab fragment,specifically binds to KIT and has a K_(D) that is more than the K_(D) ofthe native (or entire) anti-KIT antibody as assessed using methodsdescribed herein or known to one of skill in the art (e.g., a Biacore™assay, assay using KinExA 3000 instrument). In particular embodiments,an anti-KIT antibody which is an antigen-binding fragment of a whole orentire antibody, e.g., Fab fragment, specifically binds to KIT and has aK_(D) that is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, or 95% higher than the K_(D) of the whole or entireanti-KIT antibody as assessed using methods described herein or known toone of skill in the art (e.g., a Biacore™ assay, assay using KinExA 3000instrument). In particular embodiments, an anti-KIT antibody which is anantigen-binding fragment of a whole or entire antibody, e.g., Fabfragment, specifically binds to KIT and has a K_(D) that is at mostabout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, or 95% higher than the K_(D) of the whole or entire anti-KITantibody as assessed using methods described herein or known to one ofskill in the art (e.g., a Biacore™ assay, assay using KinExA 3000instrument). In particular embodiments, an anti-KIT antibody which is anantigen-binding fragment of a whole or entire antibody, e.g., Fabfragment, specifically binds to KIT and has a K_(D) that is at leastabout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, or 95% higher than the K_(D) of the whole or entire anti-KITantibody as assessed using methods described herein or known to one ofskill in the art (e.g., a Biacore™ assay, assay using KinExA 3000instrument). In particular embodiments, an anti-KIT antibody which is anantigen-binding fragment of a whole or entire antibody, e.g., Fabfragment, specifically binds to KIT and has a K_(D) that is about 1fold, 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7fold, 1.8 fold, 1.9 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold,4.5 fold or 5 fold higher than the K_(D) of the whole or entire anti-KITantibody as assessed using methods described herein or known to one ofskill in the art (e.g., a Biacore™ assay, assay using KinExA 3000instrument).

Methods for determining affinity of an antibody to its target antigenare readily available and described in the art. For example, theaffinities and binding properties of an antibody for its target antigen,can be determined by a variety of in vitro assay methods (biochemical orimmunological based assays) known in the art such as equilibrium methods(e.g., enzyme-linked immunoabsorbent assay (ELISA), or radioimmunoassay(RIA)), or kinetics (e.g., Biacore™ analysis), and other methods such asindirect binding assays, competitive inhibition assays, fluorescenceresonance energy transfer (FRET), immunoprecipitation, gelelectrophoresis and chromatography (e.g., gel filtration). These andother methods can utilize a label on one or more of the components beingexamined and/or employ a variety of detection methods including but notlimited to chromogenic, fluorescent, luminescent, or isotopic labels. Incertain embodiments, use of labels is not necessary, e.g., Biacore™systems utilize the natural phenomenon of surface plasmon resonance(SPR) to deliver data in real time, without the use of labels. Adetailed description of binding affinities and kinetics can be found inPaul, W. E., ed., Fundamental Immunology, 4th Ed. (Lippincott-Raven,Philadelphia 1999), which focuses on antibody-immunogen interactions.

In certain aspects, the affinity of an antibody described herein for aKIT antigen, e.g., human KIT, for example a D4/D5 region of KIT (e.g.,human KIT), can be characterized indirectly using cell-based assays. Forexample, cells expressing KIT on their cell membrane surface can becontacted with anti-KIT antibodies, and cellular activities downstreamof KIT can be determined using assays known in the art. For examples,phosphorylation of the cytoplasmic domain of KIT can be determined byimmunoblotting (or Western blotting) following contacting the cells withan anti-KIT antibody; cellular extracts are obtained and processed forimmunoblotting (e.g., subjecting the cellular extracts to sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferringthe proteins separated on the gel to a membrane (e.g., nitrocellulose orpolyvinylidene fluoride (PVDF)) with an antibody that specifically bindsto a phosphorylated tyrosine in the cytoplasmic domain of KIT, but doesnot bind an unphosphorylated tyrosine.

In certain embodiments, an anti-KIT antibody described hereinspecifically binds to a KIT antigen, e.g., human KIT, for example aD4/D5 region of KIT (e.g., human KIT), and induces or enhancesdimerization and phosphorylation of KIT, in the presence or absence ofthe KIT ligand SCF. In some embodiments, an anti-KIT antibody describedherein can inhibit or decrease KIT ligand, e.g., SCF, binding to KIT(i.e., an anti-KIT antibody can compete with a KIT ligand, e.g., SCF,for binding to KIT). In such case, cells can be contacted with ananti-KIT antibody and a KIT ligand, and the degree of inhibition of KITphosphorylation can be determined as an indication of the degree of theanti-KIT antibody competing with the KIT ligand for binding to KIT.

Antibodies include, but are not limited to, monoclonal antibodies,recombinantly produced antibodies, multispecific antibodies (includingbi-specific antibodies), murine antibodies, human antibodies, humanizedantibodies, chimeric antibodies, synthetic antibodies, tetramericantibodies comprising two heavy chain and two light chain molecule, anantibody light chain monomer, an antibody heavy chain monomer, anantibody light chain dimer, an antibody heavy chain dimer, an antibodylight chain-antibody heavy chain pair, intrabodies, heteroconjugateantibodies, single domain antibodies, monovalent antibodies,single-chain Fvs (scFv) (e.g., including monospecific, bispecific,etc.), camelized antibodies, affybodies, Fab fragments, F(ab′)fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id)antibodies (including, e.g., anti-anti-Id antibodies), andepitope-binding fragments of any of the above. In certain embodiments,antibodies described herein refer to polyclonal antibody populations.Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY),any class, (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ or IgA₂), or any subclass(e.g., IgG2a or IgG2b) of immunoglobulin molecule. In certainembodiments, antibodies described herein are IgG antibodies, or a class(e.g., human IgG₁ or IgG₄) or subclass thereof. In specific embodiments,a monoclonal antibody is an antibody produced by a single hybridoma orother cell, wherein the antibody immunospecifically binds to a D4/D5region of human KIT epitope as determined, e.g., by ELISA or otherantigen-binding or competitive binding assay known in the art or in theExamples provided herein. The term “monoclonal” is not limited to anyparticular method for making the antibody.

In a particular embodiment, an antibody provided herein is a Fabfragment that immunospecifically binds to a KIT polypeptide, such as theD4/D5 region of KIT. In a specific embodiment, antibodies describedherein are monoclonal antibodies or isolated monoclonal antibodies. Inanother specific embodiment, an antibody described herein is a humanizedmonoclonal antibody. In yet another specific embodiment, an antibodydescribed herein is a murine monoclonal antibody, e.g., a murinemonoclonal antibody obtained from a hybridoma. In a particularembodiment, an antibody described herein is a recombinant antibody, forexample, a recombinant human antibody or a recombinant monoclonalantibody. In certain embodiments, an antibody described herein containsnon-human amino acid sequences, e.g., non-human CDRs or non-human (e.g.,non-human primate) framework residues.

Antibodies provided herein include immunoglobulin molecules of any type(e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3,IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. In aspecific embodiment, an antibody provided herein is an IgG antibody(e.g., human IgG antibody), or a class (e.g., human IgG1 or IgG4) orsubclass thereof. In another specific embodiment, an antibody describedherein is an IgG1 (e.g., human IgG1 (isotype a, z, or f)) or IgG4antibody. In certain embodiments, an antibody described herein is awhole or entire antibody, e.g., a whole or entire humanized antibody.

Antibodies provided herein can include antibody fragments that retainthe ability to specifically bind to an epitope, i.e., KIT epitope (e.g.,a KIT epitope within a KIT polypeptide containing the D4/D5 region ofhuman KIT). In a specific embodiment, fragments include Fab fragments(an antibody fragment that contains the antigen-binding domain andcomprises a light chain and part of a heavy chain (i.e., the VH and CH1domains of a heavy chain) bridged by a disulfide bond); Fab′ (anantibody fragment containing a single antigen-binding domain comprisingan Fab and an additional portion of the heavy chain through the hingeregion); F(ab′)₂ (two Fab′ molecules joined by interchain disulfidebonds in the hinge regions of the heavy chains; the Fab′ molecules canbe directed toward the same or different epitopes); a bispecific Fab (aFab molecule having two antigen binding domains, each of which can bedirected to a different epitope); a single chain Fab chain comprising avariable region, also known as a sFv (the variable, antigen-bindingdeterminative region of a single light and heavy chain of an antibodylinked together by a chain of 10-25 amino acids); a disulfide-linked Fv,or dsFv (the variable, antigen-binding determinative region of a singlelight and heavy chain of an antibody linked together by a disulfidebond); a camelized VH (the variable, antigen-binding determinativeregion of a single heavy chain of an antibody in which some amino acidsat the VH interface are those found in the heavy chain of naturallyoccurring camel antibodies); a bispecific sFv (a sFv or a dsFv moleculehaving two antigen-binding domains, each of which can be directed to adifferent epitope); a diabody (a dimerized sFv formed when the VH domainof a first sFv assembles with the VL domain of a second sFv and the VLdomain of the first sFv assembles with the VH domain of the second sFv;the two antigen-binding regions of the diabody can be directed towardsthe same or different epitopes); and a triabody (a trimerized sFv,formed in a manner similar to a diabody, but in which threeantigen-binding domains are created in a single complex; the threeantigen binding domains can be directed towards the same or differentepitopes). Antibodies provided herein can also include one or more CDRsequences of an antibody. The CDR sequences can be linked together on ascaffold when two or more CDR sequences are present. In certainembodiments, an antibody comprises a single-chain Fv (“scFv”). scFvs areantibody fragments comprising the VH and VL domains of an antibody,wherein these domains are present in a single polypeptide chain.Generally, the scFv polypeptide further comprises a polypeptide linkerbetween the VH and VL domains which enables the scFv to form the desiredstructure for antigen binding. For a review of scFvs, see Pluckthun inThe Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Mooreeds. Springer-Verlag, New York, pp. 269-315 (1994). Without being boundby any particular theories, Fv molecules can be able to penetratetissues because of their small size. A whole antibody can beenzymatically cleaved by pepsin to produce a F(ab′)₂ fragment, or can beenzymatically cleaved by papain to produce two Fab fragments.

In certain embodiments, the antibodies described herein can be from anyanimal origin including birds (e.g., chicken or rooster) and mammals(e.g., human, mouse, donkey, sheep, rabbit, goat, guinea pig, camel,dog, cat, pig, rat, monkey, cow, hamster, or horse). In certainembodiments, the antibodies described herein are human or humanizedmonoclonal antibodies. In a certain embodiment, an antibody describedherein is a murine antibody. In a particular embodiment, an antibodydescribed herein is an antibody obtained from, or produced by, ahybridoma cell. In a particular embodiment, an antibody described hereinis an engineered antibody, for example, antibody produced by recombinantmethods. In a specific embodiment, an antibody described herein is ahumanized antibody comprising one or more non-human (e.g., rodent ormurine) CDRs and one or more human framework regions (FR), andoptionally human heavy chain constant region and/or light chain constantregion. In a specific embodiment, an antibody described herein comprisesone or more primate (or non-human primate) framework regions.

Antibodies provided herein can include antibodies comprising chemicalmodifications, for example, antibodies which have been chemicallymodified, e.g., by covalent attachment of any type of molecule to theantibody. For example, but not by way of limitation, an anti-KITantibody can be glycosylated, acetylated, pegylated, phosphorylated, oramidated, can be derivitized via protective/blocking groups, or canfurther comprise a cellular ligand and or other protein or peptide, etc.For example, an antibody provided herein can be chemically modified,e.g., by glycosylation, acetylation, pegylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, linkage to a cellular ligand or other protein,etc. Further, an anti-KIT antibody described herein can contain one ormore non-classical amino acids.

In a particular embodiment, provided herein is an anti-KIT antibodywhich has been modified in a manner suitable for large scalemanufacturing, e.g., the manufacturing platform of Lonza (Basel,Switzerland). For example, the BI-HEX® technology platform (BoehringerIngleheim, Germany) can be used to adapt the anti-KIT antibodiesdescribed herein for suitable large scale manufacturing in recombinantmammalian cell expression systems. Such adaptation can involve cloningpolynucleotide sequences encoding the necessary domains of an anti-KITantibody, such as one or more CDRs or FRs, into a suitable expressionvector which also contains polynucleotide sequences encoding suitableconstant regions, so that an entire antibody is produced. Thepolynucleotide sequences provided by the expression vectors arenucleotide sequences which can be optimized to maximize antibody yieldand stability for cell culture manufacturing conditions and purificationprocesses.

5.1.1. Conjugates

In some embodiments, provided herein are antibodies, or antigen-bindingfragments thereof, conjugated or recombinantly fused to a diagnostic,detectable or therapeutic agent or any other molecule. The conjugated orrecombinantly fused antibodies can be useful, e.g., for monitoring orprognosing the onset, development, progression and/or severity of aKIT-mediated disorder or disease, for example, as part of a clinicaltesting procedure, such as determining the efficacy of a particulartherapy. The conjugated or recombinantly fused antibodies can be useful,e.g., for treating or managing a KIT-mediated disorder (e.g., cancer),or for treating or managing effects of a KIT-mediated disorder (e.g.,cancer). Antibodies described herein can also be conjugated to amolecule (e.g., polyethylene glycol) which can affect one or morebiological and/or molecular properties of the antibodies, for example,stability (e.g., in serum), half-life, solubility, and antigenicity.

In a particular aspect, provided herein is a conjugate comprising anagent (e.g., therapeutic agent) linked to an antibody described herein(or an antigen-binding fragment thereof), which antibodyimmunospecifically binds to a D4/D5 region of human KIT (e.g., SEQ IDNO: 15). In a specific embodiment, the conjugated antibody specificallybinds a D4/D5 region of KIT (e.g., human KIT), and comprises antibody37M or 37C, a KIT-binding portion thereof, e.g., any such portiondescribed herein, or an antibody comprising the CDRs (e.g., 3 VL CDRsand/or 3 VH CDRS) of antibody 37M or 37C. In a specific embodiment, aconjugated antibody specifically binds a D4/D5 region of KIT (e.g.,human KIT) and comprises a VL chain region comprising VL CDR1, VL CDR2,and VL CDR3 having the amino acid sequence of SEQ ID NO: 20, 21, and 22,respectively, and/or a VH chain region comprising VH CDR1, VH CDR2, andVH CDR3 having the amino acid sequence of SEQ ID NO: 23, 24, and 25,respectively. In one embodiment, an antibody that is conjugated is onethat binds a D4/D5 region of human KIT with an affinity of less thanabout 200 pM. In another embodiment, an antibody that is conjugated isone that inhibits a biological activity of KIT. In specific embodiments,a conjugate comprises an antibody described herein and a molecule (e.g.,therapeutic or drug moiety), wherein the antibody is linked directly tothe molecule, or by way of one or more linkers. In certain embodiments,an antibody is covalently conjugated to a molecule. In a particularembodiment, an antibody is noncovalently conjugated to a molecule. Inspecific embodiments, an antibody described herein, e.g., an antibodyconjugated to an agent, binds to wild-type human KIT. In certainembodiments, an antibody described herein, e.g., antibody conjugated toan agent, binds to an extracellular domain of human KIT comprising amutation, for example a somatic mutation associated with cancer (e.g.,GIST), such as a mutation in exon 9 of human KIT wherein the Ala and Tyrresidues at positions 502 and 503 are duplicated.

Such diagnosis and detection can be accomplished, for example, bycoupling the antibody to detectable molecules or substances including,but not limited to, various enzymes, such as, but not limited to,horseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; prosthetic groups, such as, but not limited to,streptavidin/biotin and avidin/biotin; fluorescent materials, such as,but not limited to, umbelliferone, fluorescein, fluoresceinisothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; luminescent materials, such as, but notlimited to, luminol; bioluminescent materials, such as but not limitedto, luciferase, luciferin, and aequorin; radioactive materials, such as,but not limited to, iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon (¹⁴C),sulfur (³⁵S), tritium (³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In, and ¹¹¹In),technetium (⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium(¹⁰³Pd) molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu,¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr,¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn,⁷⁵Se, ¹¹³Sn, and ¹¹⁷Sn; and positron emitting metals using variouspositron emission tomographies, and non-radioactive paramagnetic metalions.

Provided are antibodies described herein, or antigen-binding fragmentsthereof, conjugated or recombinantly fused to a therapeutic moiety (orone or more therapeutic moieties) and uses of such antibodies. Theantibody can be conjugated or recombinantly fused to a therapeuticmoiety, such as a cytotoxin, e.g., a cytostatic or cytocidal agent, atherapeutic agent or a radioactive metal ion, e.g., alpha-emitters. Acytotoxin or cytotoxic agent includes any agent that is detrimental tocells. Therapeutic moieties include, but are not limited to, auristatinor a derivative thereof, e.g., monomethyl auristatin E (MMAE),monomethyl auristatin F (MMAF), auristatin PYE, and auristatin E (AE)(see, e.g., U.S. Pat. No. 7,662,387 and U.S. Pat. ApplicationPublication Nos. 2008/0300192 and 2008/0025989); amicrotubule-disrupting agent, e.g., maytansine or a derivative thereof,e.g., maytansinoid DM1 (see, e.g., U.S. Pat. Nos. 7,851,432, 7,575,748,and 5,416,064); a prodrug, e.g., a prodrug of a CC-1065 (rachelmycin)analogue; antimetabolites (e.g., methotrexate, 6-mercaptopurine,6-thioguanine, cytarabine, 5-fluorouracil decarbazine); alkylatingagents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,carmustine (BCNU) and lomustine (CCNU), cyclothosphamide, busulfan,dibromomannitol, streptozotocin, mitomycin C, and cisdichlorodiamineplatinum (II) (DDP), and cisplatin); minor-groove-binding alkylatingagent; anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin); antibiotics (e.g., d actinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)); Auristatin molecules(e.g., auristatin PHE, bryostatin 1, and solastatin 10; see Woyke etal., Antimicrob. Agents Chemother. 46:3802-8 (2002), Woyke et al.,Antimicrob. Agents Chemother. 45:3580-4 (2001), Mohammad et al.,Anticancer Drugs 12:735-40 (2001), Wall et al., Biochem. Biophys. Res.Commun. 266:76-80 (1999), Mohammad et al., Int. J. Oncol. 15:367-72(1999), all of which are incorporated herein by reference); hormones(e.g., glucocorticoids, progestins, androgens, and estrogens),DNA-repair enzyme inhibitors (e.g., etoposide or topotecan), kinaseinhibitors (e.g., compound ST1571, imatinib mesylate (Kantarjian et al.,Clin Cancer Res. 8(7):2167-76 (2002)); cytotoxic agents (e.g.,paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof and those compounds disclosed in U.S. Pat. Nos.6,245,759, 6,399,633, 6,383,790, 6,335,156, 6,271,242, 6,242,196,6,218,410, 6,218,372, 6,057,300, 6,034,053, 5,985,877, 5,958,769,5,925,376, 5,922,844, 5,911,995, 5,872,223, 5,863,904, 5,840,745,5,728,868, 5,648,239, 5,587,459); farnesyl transferase inhibitors (e.g.,R115777, BMS-214662, and those disclosed by, for example, U.S. Pat. Nos.6,458,935, 6,451,812, 6,440,974, 6,436,960, 6,432,959, 6,420,387,6,414,145, 6,410,541, 6,410,539, 6,403,581, 6,399,615, 6,387,905,6,372,747, 6,369,034, 6,362,188, 6,342,765, 6,342,487, 6,300,501,6,268,363, 6,265,422, 6,248,756, 6,239,140, 6,232,338, 6,228,865,6,228,856, 6,225,322, 6,218,406, 6,211,193, 6,187,786, 6,169,096,6,159,984, 6,143,766, 6,133,303, 6,127,366, 6,124,465, 6,124,295,6,103,723, 6,093,737, 6,090,948, 6,080,870, 6,077,853, 6,071,935,6,066,738, 6,063,930, 6,054,466, 6,051,582, 6,051,574, and 6,040,305);topoisomerase inhibitors (e.g., camptothecin; irinotecan; SN-38;topotecan; 9-aminocamptothecin; GG-211 (GI 147211); DX-8951f; IST-622;rubitecan; pyrazoloacridine; XR-5000; saintopin; UCE6; UCE1022;TAN-1518A; TAN 1518B; KT6006; KT6528; ED-110; NB-506; ED-110; NB-506;and rebeccamycin); bulgarein; DNA minor groove binders such as Hoeschtdye 33342 and Hoechst dye 33258; nitidine; fagaronine; epiberberine;coralyne; beta-lapachone; BC-4-1; bisphosphonates (e.g., alendronate,cimadronte, clodronate, tiludronate, etidronate, ibandronate,neridronate, olpandronate, risedronate, piridronate, pamidronate,zolendronate) HMG-CoA reductase inhibitors, (e.g., lovastatin,simvastatin, atorvastatin, pravastatin, fluvastatin, statin,cerivastatin, lescol, lupitor, rosuvastatin and atorvastatin); antisenseoligonucleotides (e.g., those disclosed in the U.S. Pat. Nos. 6,277,832,5,998,596, 5,885,834, 5,734,033, and 5,618,709); adenosine deaminaseinhibitors (e.g., Fludarabine phosphate and 2-Chlorodeoxyadenosine);ibritumomab tiuxetan (Zevalin®); tositumomab (Bexxar®)) andpharmaceutically acceptable salts, solvates, clathrates, and prodrugsthereof. In one embodiment, an antibody that is conjugated to suchtherapeutic/drug moiety is one that binds a D4/D5 region of human KITwith an affinity of less than about 200 pM. In another embodiment, anantibody that is conjugated to such therapeutic/drug moiety is one thatinhibits a biological activity of KIT. In a specific embodiment, anantibody that is conjugated to such therapeutic/drug moiety is one thatcomprises CDRs of antibody 37M or 37C (e.g., VL CDR1, VL CDR2, and VLCDR3 having the amino acid sequence of SEQ ID NO: 20, 21, and 22,respectively, and/or a VH chain region comprising VH CDR1, VH CDR2, andVH CDR3 having the amino acid sequence of SEQ ID NO: 23, 24, and 25,respectively).

In particular embodiments, a therapeutic moiety or drug moiety is anantitubulin drug, such as an auristatin or a derivative thereof.Non-limiting examples of auristatins include monomethyl auristatin E(MMAE), monomethyl auristatin F (MMAF), auristatin PYE, and auristatin E(AE) (see, e.g., U.S. Pat. No. 7,662,387 and U.S. Pat. ApplicationPublication Nos. 2008/0300192 and 2008/0025989). In certain embodiments,a therapeutic moiety or drug moiety is a microtubule-disrupting agentsuch as maytansine or a derivative thereof, e.g., maytansinoid DM1 orDM4 (see, e.g., U.S. Pat. Nos. 7,851,432, 7,575,748, and 5,416,064). Incertain embodiments, a therapeutic moiety or drug moiety is a prodrug,e.g., a prodrug of a CC-1065 (rachelmycin) analogue (see, e.g., U.S.Patent Application Publication No. 2008/0279868, and PCT InternationalPatent Application Publication Nos. WO 2009/017394, WO 2010/062171, andWO 2007/089149). In one embodiment, an antibody that is conjugated tosuch therapeutic/drug moiety is one that binds a D4/D5 region of humanKIT with an affinity of less than about 200 pM. In another embodiment,an antibody that is conjugated to such therapeutic/drug moiety is onethat inhibits a biological activity of KIT. In a specific embodiment, anantibody that is conjugated to such therapeutic/drug moiety is one thatcomprises CDRs of antibody 37M or 37C (e.g., VL CDR1, VL CDR2, and VLCDR3 having the amino acid sequence of SEQ ID NO: 20, 21, and 22,respectively, and/or a VH chain region comprising VH CDR1, VH CDR2, andVH CDR3 having the amino acid sequence of SEQ ID NO: 23, 24, and 25,respectively).

In a specific embodiment, the antibody and therapeutic/drug agent areconjugated by way of one or more linkers. In another specificembodiment, the antibody and therapeutic/drug agent are conjugateddirectly.

In specific embodiments, non-limiting examples of therapeutic moietiesor drug moieties for conjugation to an antibody described herein includecalicheamicins (e.g., LL-E33288 complex, for example,gamma-calicheamicin, see, e.g., U.S. Pat. No. 4,970,198) and derivativesthereof (e.g., gamma calicheamicin hydrazide derivatives), duocarmycinsand derivatives thereof (e.g., CC-1065 (NSC 298223), or an achiralanalogue of duocarmycin (for example AS-1-145 or centanamycin)), taxanesand derivatives thereof, and enediynes and derivatives thereof (See,e.g., PCT International Patent Application Publication Nos. WO2009/017394, WO 2010/062171, WO 2007/089149, WO 2011/021146, WO2008/150261, WO 2006/031653, WO 2005/089809, WO 2005/089807, and WO2005/089808, each of which is incorporated by reference herein in itsentirety). In a specific embodiment, an antibody that is conjugated tosuch therapeutic/drug moiety is one that comprises CDRs of antibody 37Mor 37C (e.g., VL CDR1, VL CDR2, and VL CDR3 having the amino acidsequence of SEQ ID NO: 20, 21, and 22, respectively, and/or a VH chainregion comprising VH CDR1, VH CDR2, and VH CDR3 having the amino acidsequence of SEQ ID NO: 23, 24, and 25, respectively). In a specificembodiment, the antibody and therapeutic agent are conjugated by way ofone or more linkers. In another specific embodiment, the antibody andtherapeutic agent are conjugated directly.

Non-limiting examples of calicheamicins suitable for conjugation to anantibody described herein are disclosed, for example, in U.S. Pat. Nos.4,671,958; 5,053,394; 5,037,651; 5,079,233; and 5,108,912; and PCTInternational Patent Application Publication Nos. WO 2011/021146, WO2008/150261, WO 2006/031653, WO 2005/089809, WO 2005/089807, and WO2005/089808; which are incorporated by reference herein in theirentirety. In particular embodiments, these compounds may contain amethyltrisulfide that reacts with appropriate thiols to form disulfides,and at the same time introduces a functional group such as a hydrazideor other functional group that may be useful for conjugatingcalicheamicin to an antibody described herein. In certain embodiments,stabilizing the disulfide bond that is present in calicheamicinconjugates by adding dimethyl substituents may yield an improvedantibody/drug conjugate. In specific embodiments, the calicheamicinderivative is N-acetyl gamma calicheamicin dimethyl hydrazide, orNAc-gamma DMH (CL-184,538), as one of the optimized derivatives forconjugation. Disulfide analogs of calicheamicin which can be conjugatedto the antibody described herein are described, for example, in U.S.Pat. Nos. 5,606,040 and 5,770,710, which are incorporated by referenceherein in their entirety. In a certain embodiment, a moiety (e.g.,calicheamicin or a derivative thereof) is conjugated to an antibody by alinker. In a particular embodiment, a moiety (e.g., calicheamicin or aderivative thereof) is hydrolyzed from the antibody-drug conjugate atthe linker. In one embodiment, a moiety (e.g., calicheamicin or aderivative thereof) is hydrolyzed from an antibody conjugate at thelinker between about a pH of 3.0 and pH 4.0 for 1-24 hours at atemperature from 20 to 50° C., preferably 37° C.

In specific embodiments, non-limiting examples of therapeutic moietiesor drug moieties for conjugation to an antibody described herein includepyrrolobenzodiazepines (PBDs) and derivatives thereof, for example, PBDdimers (e.g., SJG-136 or SG2000), C2-unsaturated PBD dimers,pyrrolobenzodiazepine dimers bearing C2 aryl substitutions (e.g.,SG2285), PBD dimer pro-drug that is activated by hydrolysis (e.g.,SG2285), and polypyrrole-PBD (e.g., SG2274) (see, e.g., PCTInternational Patent Application Publication Nos. WO 2000/012507, WO2007/039752, WO 2005/110423, WO 2005/085251, and WO 2005/040170, andU.S. Pat. No. 7,612,062, each of which is incorporated by referenceherein in its entirety). In a specific embodiment, an antibody that isconjugated to such therapeutic/drug moiety is one that comprises CDRs ofantibody 37M or 37C (e.g., VL CDR1, VL CDR2, and VL CDR3 having theamino acid sequence of SEQ ID NO: 20, 21, and 22, respectively, and/or aVH chain region comprising VH CDR1, VH CDR2, and VH CDR3 having theamino acid sequence of SEQ ID NO: 23, 24, and 25, respectively). In aspecific embodiment, the antibody and therapeutic agent is conjugated byway of one or more linkers.

Further, an antibody described herein can be conjugated or recombinantlyfused to a therapeutic moiety or drug moiety that modifies a givenbiological response. Therapeutic moieties or drug moieties are not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety can be a protein, peptide, or polypeptidepossessing a desired biological activity. Such proteins can include, forexample, a toxin such as abrin, ricin A, pseudomonas exotoxin, choleratoxin, or diphtheria toxin; a protein such as tumor necrosis factor,γ-interferon, α-interferon, nerve growth factor, platelet derived growthfactor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-γ,TNF-γ, AIM I (see, International Publication No. WO 97/33899), AIM II(see, International Publication No. WO 97/34911), Fas Ligand (Takahashiet al., 1994, J. Immunol., 6:1567-1574), and VEGF (see, InternationalPublication No. WO 99/23105), an anti-angiogenic agent, e.g.,angiostatin, endostatin or a component of the coagulation pathway (e.g.,tissue factor); or, a biological response modifier such as, for example,a lymphokine (e.g., interferon gamma, interleukin-1 (“IL-1”),interleukin-2 (“IL-2”), interleukin-5 (“IL-5”), interleukin-6 (“IL-6”),interleukin-7 (“IL-7”), interleukin 9 (“IL-9”), interleukin-10(“IL-10”), interleukin-12 (“IL-12”), interleukin-15 (“IL-15”),interleukin-23 (“IL-23”), granulocyte macrophage colony stimulatingfactor (“GM-CSF”), and granulocyte colony stimulating factor (“G-CSF”)),or a growth factor (e.g., growth hormone (“GH”)), or a coagulation agent(e.g., calcium, vitamin K, tissue factors, such as but not limited to,Hageman factor (factor XII), high-molecular-weight kininogen (HMWK),prekallikrein (PK), coagulation proteins-factors II (prothrombin),factor V, XIIa, VIII, XIIIa, XI, XIa, IX, IXa, X, phospholipid, andfibrin monomer).

Provided herein are antibodies recombinantly fused or chemicallyconjugated (covalent or non-covalent conjugations) to a heterologousprotein or polypeptide (or fragment thereof, preferably to a polypeptideof about 10, about 20, about 30, about 40, about 50, about 60, about 70,about 80, about 90 or about 100 amino acids) to generate fusionproteins. In particular, provided herein are fusion proteins comprisingan antigen-binding fragment of an antibody described herein (e.g., a Fabfragment, Fd fragment, Fv fragment, F(ab)₂ fragment, a VH domain, a VHCDR, a VL domain or a VL CDR) and a heterologous protein, polypeptide,or peptide. In one embodiment, the heterologous protein, polypeptide, orpeptide that the antibody is fused to is useful for targeting theantibody to a particular cell type, such as a cell that expresses KIT.For example, an antibody that immunospecifically binds to a cell surfacereceptor expressed by a particular cell type (e.g., an immune cell) canbe fused or conjugated to a modified antibody described herein. Inspecific embodiments, the heterologous protein or polypeptide (orfragment thereof) binds to a second target (e.g., a target other thanKIT) (see, e.g., PCT International Patent Application Publication No. WO2009/088805 and U.S. Patent Application Publication No. US2009/0148905).

Provided herein is a conjugated or fusion protein comprising anyantibody described herein, or an antigen-binding fragment thereof, and aheterologous polypeptide (e.g., a polypeptide other than KIT). In oneembodiment, a conjugated or fusion protein described herein comprises ananti-KIT antibody described herein, and a heterologous polypeptide. Inanother embodiment, a conjugated or fusion protein provided hereincomprises an antigen-binding fragment of an anti-KIT antibody describedherein, and a heterologous polypeptide. In another embodiment, aconjugated or fusion protein described herein comprises a VH domainhaving the amino acid sequence of any one of the VH domains of ananti-KIT antibody described herein, and/or a VL domain having the aminoacid sequence of any one of the VL domains of an anti-KIT antibodydescribed herein, and a heterologous polypeptide. In another embodiment,a conjugated or fusion protein described herein comprises one or more VHCDRs having the amino acid sequence of any one of SEQ ID NO: 23, 24, and25, and a heterologous polypeptide. In another embodiment, a conjugatedor fusion protein comprises one or more VL CDRs having the amino acidsequence of any one of the VL CDRs of an anti-KIT antibody describedherein (e.g., VL CDRs in Table, SEQ ID NOs: 21, 22, and 23), and aheterologous polypeptide. In another embodiment, a conjugated or fusionprotein described herein comprises at least one VH domain and at leastone VL domain of an anti-KIT antibody described herein, and aheterologous polypeptide. In yet another embodiment, a conjugated orfusion protein described herein comprises at least one VH CDR and atleast one VL CDR of an anti-KIT antibody described herein (e.g., VL CDRsin Table 1 and VH CDRs in Table 3), and a heterologous polypeptide.

In addition, an antibody described herein can be conjugated totherapeutic moieties such as a radioactive metal ion, such asalpha-emitters such as ²¹³Bi or macrocyclic chelators useful forconjugating radiometal ions, including but not limited to, ¹³¹In, ¹³¹LU,¹³¹Y, ¹³¹Ho, ¹³¹Sm, to polypeptides. In certain embodiments, themacrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) whichcan be attached to the antibody via a linker molecule. Such linkermolecules are commonly known in the art and described in Denardo et al.,1998, Clin Cancer Res. 4(10):2483-90; Peterson et al., 1999, Bioconjug.Chem. 10(4):553-7; and Zimmerman et al., 1999, Nucl. Med. Biol.26(8):943-50, each incorporated by reference in their entireties.

In certain embodiments, an antibody described herein, or anantigen-binding fragment thereof, is conjugated to one or more molecules(e.g., therapeutic or drug moiety) directly or indirectly via one ormore linker molecules. In particular embodiments, a linker is anenzyme-cleavable linker or a disulfide linker. In a specific embodiment,the cleavable linker is cleavable via an enzyme such an aminopeptidase,an aminoesterase, a dipeptidyl carboxy peptidase, or a protease of theblood clotting cascade. In particular embodiments, a linker comprises 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20 amino acidresidues. In certain embodiments, a linker consists of 1 to 10 aminoacid residues, 1 to 15 amino acid residues, 5 to 20 amino acid residues,10 to 25 amino acid residues, 10 to 30 amino acid residues, or 10 to 50amino acid residues.

In certain embodiments, a moiety is conjugated to an antibody by one ormore linkers. In a particular embodiment, a moiety is hydrolyzed fromthe antibody-drug conjugate at the linker. In one embodiment, a moietyis hydrolyzed from the antibody conjugate at the linker between about apH of 3.0 and pH 4.0 for about 1-24 hours, and at a temperature fromabout 20 to 50° C., preferably 37° C. In a specific embodiment, a linkeris stable in the blood stream but releases the conjugated moiety once itis inside the targeted cells. In certain embodiments, a moiety isconjugated to an antibody described herein via one or moretriazole-containing linkers (see, e.g., International Patent ApplicationPublication No. WO 2007/018431, which is incorporated by referenceherein in its entirety). Non-limiting examples of linkers and spacersfor incorporation into antibody-drug conjugates described herein aredisclosed in PCT International Patent Application Publication Nos. WO2007/018431, WO 2004/043493, and WO 2002/083180.

Moreover, antibodies described herein can be fused to marker sequences,such as a peptide to facilitate purification. In preferred embodiments,the marker amino acid sequence is a hexa-histidine peptide, such as thetag provided in a pQE vector (QIAGEN, Inc.), among others, many of whichare commercially available. As described in Gentz et al., 1989, Proc.Natl. Acad. Sci. USA 86:821-824, for instance, hexa-histidine providesfor convenient purification of the fusion protein. Other peptide tagsuseful for purification include, but are not limited to, thehemagglutinin (“HA”) tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767),and the “FLAG” tag.

Methods for fusing or conjugating therapeutic moieties (includingpolypeptides) to antibodies are well known, see, e.g., Arnon et al.,“Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”,in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp.243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For DrugDelivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al.(eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “AntibodyCarriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in MonoclonalAntibodies 84: Biological And Clinical Applications, Pinchera et al.(eds.), pp. 475-506 (1985); “Analysis, Results, And Future ProspectiveOf The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), Thorpe et al., 1982, Immunol.Rev. 62:119-58; U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046,5,349,053, 5,447,851, 5,723,125, 5,783,181, 5,908,626, 5,844,095, and5,112,946; EP 307,434; EP 367,166; EP 394,827; PCT publications WO91/06570, WO 96/04388, WO 96/22024, WO 97/34631, and WO 99/04813;Ashkenazi et al., Proc. Natl. Acad. Sci. USA, 88: 10535-10539, 1991;Traunecker et al., Nature, 331:84-86, 1988; Zheng et al., J. Immunol.,154:5590-5600, 1995; Vil et al., Proc. Natl. Acad. Sci. USA,89:11337-11341, 1992, which are incorporated herein by reference intheir entireties.

Fusion proteins can be generated, for example, through the techniques ofgene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling(collectively referred to as “DNA shuffling”). DNA shuffling can beemployed to alter the activities of antibodies described herein (e.g.,antibodies with higher affinities and lower dissociation rates). See,generally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252,and 5,837,458; Patten et al., 1997, Curr. Opinion Biotechnol. 8:724-33;Harayama, 1998, Trends Biotechnol. 16(2):76-82; Hansson et al., 1999, J.Mol. Biol. 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques24(2):308-313 (each of these patents and publications are herebyincorporated by reference in its entirety). Antibodies, or the encodedantibodies, can be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. A polynucleotide encoding an antibody described hereincan be recombined with one or more components, motifs, sections, parts,domains, fragments, etc. of one or more heterologous molecules.

An antibody described herein can also be conjugated to a second antibodyto form an antibody heteroconjugate as described in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

The therapeutic moiety or drug conjugated or recombinantly fused to anantibody described herein that immunospecifically binds to a KIT antigencan be chosen to achieve the desired prophylactic or therapeuticeffect(s), e.g., reducing tumor size or burden, reducing cancer cellgrowth or proliferation, or inducing death of cancer cells. In certainembodiments, the antibody is a modified antibody. A clinician or othermedical personnel should consider the following when deciding on whichtherapeutic moiety or drug to conjugate or recombinantly fuse to anantibody described herein: the nature of the disease, the severity ofthe disease, and the condition of the subject.

Antibodies described herein can also be attached to solid supports,which are particularly useful for immunoassays or purification of thetarget antigen. Such solid supports include, but are not limited to,glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chlorideor polypropylene.

5.2 Polynucleotides

In certain aspects, provided herein are polynucleotides comprising anucleotide sequence encoding an antibody described herein or a fragmentthereof (e.g., a variable light chain region and/or variable heavy chainregion) that immunospecifically binds to a KIT antigen, and vectors,e.g., vectors comprising such polynucleotides for recombinant expressionin host cells (e.g., E. coli and mammalian cells). Provided herein arepolynucleotides comprising nucleotide sequences encoding any of theantibodies provided herein, as well as vectors comprising suchpolynucleotide sequences, e.g., expression vectors for their efficientexpression in host cells, e.g., mammalian cells. Also provided hereinare polynucleotides encoding KIT antigens (e.g., SEQ ID NO: 14 or 15)for generating anti-KIT antibodies described herein.

In particular aspects, provided herein are polynucleotides comprisingnucleotide sequences encoding antibodies (e.g., a murine, chimeric, orhumanized antibody) or antigen-binding fragments thereof, whichimmunospecifically bind to a KIT polypeptide (e.g., the D4/D5 region ofKIT, for example, human KIT) and comprises an amino acid sequence asdescribed herein, as well as antibodies which compete with suchantibodies for binding to a KIT polypeptide (e.g., in a dose-dependentmanner), or which binds to the same epitope as that of such antibodies.

In certain aspects, provided herein are polynucleotides comprising anucleotide sequence encoding the light chain or heavy chain of anantibody described herein. The polynucleotides can comprise nucleotidesequences encoding a light chain comprising the VL FRs and CDRs ofantibodies described herein (see, e.g., Tables 1 and 2). Thepolynucleotides can comprise nucleotide sequences encoding a heavy chaincomprising the VH FRs and CDRs of antibodies described herein (see,e.g., Tables 3 and 4). In specific embodiments, a polynucleotidedescribed herein encodes a VL chain region having the amino acidsequence of SEQ ID NO: 2. In specific embodiments, a polynucleotidedescribed herein encodes a VH chain region having the amino acidsequence of any one of SEQ ID NOs: 3 and 5. In particular embodiments, apolynucleotide described herein encodes a VL chain region having theamino acid sequence of SEQ ID NO: 2. In particular embodiments, apolynucleotide described herein encodes a VH chain region having theamino acid sequence of SEQ ID NO: 3. In particular embodiments, apolynucleotide described herein encodes a VH chain region having theamino acid sequence of SEQ ID NO: 5.

In particular embodiments, a polynucleotide described herein encodes aVL chain region, wherein the polynucleotide comprises the nucleic acidsequence of SEQ ID NO: 8. In particular embodiments, a polynucleotidedescribed herein encodes a VH chain region, wherein the polynucleotidecomprises the nucleic acid sequence of SEQ ID NO: 9. In particularembodiments, a polynucleotide encodes an antibody described herein,wherein the polynucleotide comprises the nucleic acid sequence of SEQ IDNO: 8 encoding a VL chain region and the nucleic acid sequence of SEQ IDNO: 9 encoding a VH chain region. In particular embodiments, one or morepolynucleotides comprise the nucleic acid sequence of SEQ ID NO: 8encoding a VL chain region and the nucleic acid sequence of SEQ ID NO: 9encoding a VH chain region. In particular embodiments, a polynucleotidedescribed herein encodes a VL chain region, wherein the polynucleotidecomprises a nucleic acid sequence that is at least 80%, at least 85%, atleast 90%, at least 95%, or at least 98% identical to the nucleic acidsequence of SEQ ID NO: 8. In particular embodiments, a polynucleotidedescribed herein encodes a VH chain region, wherein the polynucleotidecomprises a nucleic acid sequence that is at least 80%, at least 85%, atleast 90%, at least 95%, or at least 98% identical to the nucleic acidsequence of SEQ ID NO: 9.

In particular embodiments, a polynucleotide described herein encodes alight chain, wherein the polynucleotide comprises the nucleic acidsequence of SEQ ID NO: 10. In a particular embodiment, a polynucleotidedescribed herein encodes a heavy chain, wherein the polynucleotidecomprises the nucleic acid sequence of SEQ ID NO: 11. In a certainembodiment, a polynucleotide comprises the nucleic acid sequence of SEQID NO: 10 encoding a light chain and the nucleic acid sequence of SEQ IDNO: 11 encoding a heavy chain. In a certain embodiment, one or morepolynucleotides comprise the nucleic acid sequence of SEQ ID NO: 10encoding a light chain and the nucleic acid sequence of SEQ ID NO: 11encoding a heavy chain. In particular embodiments, a polynucleotidedescribed herein encodes a light chain, wherein the polynucleotidecomprises a nucleic acid sequence that is at least 80%, at least 85%, atleast 90%, at least 95%, or at least 98% identical to the nucleic acidsequence of SEQ ID NO: 10. In particular embodiments, a polynucleotidedescribed herein encodes a heavy chain, wherein the polynucleotidecomprises a nucleic acid sequence that is at least 80%, at least 85%, atleast 90%, at least 95%, or at least 98% identical to the nucleic acidsequence of SEQ ID NO: 11.

In particular embodiments, provided herein are polynucleotidescomprising a nucleotide sequence encoding an anti-KIT antibodycomprising a VL chain region (e.g., containingFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4) having the amino acid sequencesdescribed herein (e.g., see Tables 1 and 2). In specific embodiments,provided herein are polynucleotides comprising a nucleotide sequenceencoding an anti-KIT antibody comprising a VH chain region (e.g.,containing FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4) having the amino acidsequence described herein (e.g., see Tables 3 and 4).

In a particular embodiment, a polynucleotide described herein comprisesa nucleotide sequence encoding an antibody provided herein comprising aVL chain region having the amino acid sequence of SEQ ID NO: 2, and a VHchain region having the amino acid sequence of SEQ ID NO: 3. In aparticular embodiment, a polynucleotide described herein comprises anucleotide sequence encoding an antibody provided herein comprising a VLchain region having the amino acid sequence of SEQ ID NO: 2, and a VHchain region having the amino acid sequence of SEQ ID NO: 5.

In a particular embodiment, a polynucleotide described herein comprisesa nucleotide sequence encoding an antibody provided herein comprising alight chain comprising the amino acid sequence of SEQ ID NO: 6 (or theamino acid sequence of SEQ ID NO: 6 starting at position 20, lacking thesignal peptide), and a heavy chain comprising the amino acid sequence ofSEQ ID NO: 7 (or the amino acid sequence of SEQ ID NO: 7 starting atposition 20, lacking the signal peptide).

In certain embodiments, a polynucleotide described herein comprises anucleotide sequence encoding an antibody provided herein comprising avariable light (VL) chain region comprising an amino acid describedherein (e.g., see FIG. 3A), wherein the antibody immunospecificallybinds to a KIT polypeptide, e.g., a human KIT polypeptide, for example,a D4/D5 region of KIT (e.g., human KIT), for example SEQ ID NO: 15.

In certain embodiments, a polynucleotide described herein comprises anucleotide sequence encoding an antibody provided herein comprising avariable heavy (VH) chain region comprising an amino acid sequencedescribed herein (e.g., see FIG. 3B), wherein the antibodyimmunospecifically binds to a KIT polypeptide, e.g., a human KITpolypeptide, for example, a D4/D5 region of KIT (e.g., human KIT), forexample SEQ ID NO: 15.

In certain embodiments, a polynucleotide described herein comprises anucleotide sequence encoding an antibody provided herein comprises oneor more VL CDRs having the amino acid sequence described herein (e.g.,see Table 1), wherein the antibody immunospecifically binds to a KITpolypeptide, e.g., a human KIT polypeptide, for example, a D4/D5 regionof KIT (e.g., human KIT), for example SEQ ID NO: 15.

In certain embodiments, a polynucleotide described herein comprises anucleotide sequence encoding an antibody provided herein comprising oneor more VH CDRs having the amino acid sequence described herein (e.g.,see Table 3), wherein the antibody immunospecifically binds to a KITpolypeptide, e.g., a human KIT polypeptide, for example, a D4/D5 regionof KIT (e.g., human KIT), for example SEQ ID NO: 15.

In another specific embodiment, a polynucleotide described hereincomprises a nucleotide sequence encoding an antibody provided herein(e.g., murine, chimeric, or humanized antibody) comprising: (i) a VLchain region comprising a VL chain region comprising a VL CDR1, VL CDR2,and VL CDR3 having the amino acid sequences of SEQ ID NOs: 20, 21, and22, respectively; and (ii) a VH chain region comprising a VH CDR1, VHCDR2, and VH CDR3 having the amino acid sequences of SEQ ID NOs: 23, 24,and 25, respectively.

In specific aspects, provided herein is a polynucleotide describedherein comprises a nucleotide sequence encoding an antibody providedherein (e.g., murine, chimeric, or humanized antibody) whichcompetitively blocks (e.g., in a dose dependent manner), antibodiescomprising the amino acid sequences described herein from specificbinding to a KIT polypeptide (e.g., a D4/D5 region of KIT, for examplehuman KIT), as determined using assays known to one of skill in the artor described herein (e.g., ELISA competitive assays).

In certain aspects, a polynucleotide comprises a nucleotide sequenceencoding an antibody described herein comprising a VL chain regioncomprising one or more VL FRs having the amino acid sequence describedherein (e.g., see Table 2), wherein the antibody immunospecificallybinds to a KIT polypeptide, e.g., a human KIT polypeptide, for example,a D4/D5 region of KIT (e.g., human KIT), for example SEQ ID NO: 15. Incertain aspects, a polynucleotide comprises a nucleotide sequenceencoding an antibody described herein comprising a VH chain regioncomprising one or more VH FRs having the amino acid sequence describedherein (e.g., see Table 4), wherein the antibody immunospecificallybinds to a KIT polypeptide, e.g., a human KIT polypeptide, for example,a D4/D5 region of KIT (e.g., human KIT), for example SEQ ID NO: 15.

In specific embodiments, a polynucleotide provided herein comprises anucleotide sequence encoding an antibody described herein comprising:framework regions (e.g., framework regions of the VL domain and VHdomain) that are human framework regions, wherein the immunospecificallybinds to a KIT polypeptide, e.g., a human KIT polypeptide, for example,a D4/D5 region of KIT (e.g., human KIT, for example SEQ ID NO: 15).

In specific aspects, provided herein is a polynucleotide comprising anucleotide sequence encoding an antibody comprising a light chain and aheavy chain, e.g., a separate light chain and heavy chain. With respectto the light chain, in a specific embodiment, a polynucleotide providedherein comprises a nucleotide sequence encoding a kappa light chain. Inanother specific embodiment, a polynucleotide provided herein comprisesa nucleotide sequence encoding a lambda light chain. In yet anotherspecific embodiment, a polynucleotide provided herein comprises anucleotide sequence encoding an antibody described herein comprising ahuman kappa light chain or a human lambda light chain. In a particularembodiment, a polynucleotide provided herein comprises a nucleotidesequence encoding an antibody described herein, which immunospecificallybinds to a KIT polypeptide (e.g., a KIT polypeptide comprising a D4/D5region of KIT, for example human KIT (e.g., SEQ ID NO: 15)), wherein theantibody comprises a light chain, and wherein the amino acid sequence ofthe VL chain region can comprise any amino acid sequence describedherein (e.g., SEQ ID NO: 2), and wherein the constant region of thelight chain comprises the amino acid sequence of a human kappa lightchain constant region. In a particular embodiment, the light chaincomprises the amino acid sequence of SEQ ID NO: 12. In anotherparticular embodiment, a polynucleotide provided herein comprises anucleotide sequence encoding an antibody described herein, whichimmunospecifically binds to a KIT polypeptide (e.g., a KIT polypeptidecomprising a KIT polypeptide comprising a D4/D5 region of KIT, forexample human KIT (e.g., SEQ ID NO: 15)), and comprises a light chain,wherein the amino acid sequence of the VL chain region can comprises anyamino acid sequence described herein (e.g., SEQ ID NO: 3 or 5), andwherein the constant region of the light chain comprises the amino acidsequence of a human lambda light chain constant region. For example,human constant region sequences can be those described in U.S. Pat. No.5,693,780. In certain embodiments, the constant region of the lightchain comprises the amino acid sequence of SEQ ID NO: 12.

In a particular embodiment, a polynucleotide provided herein comprises anucleotide sequence encoding an antibody described herein, whichimmunospecifically binds to a KIT polypeptide (e.g., a KIT polypeptidecomprising a KIT polypeptide comprising a D4/D5 region of KIT, forexample human KIT (e.g., SEQ ID NO: 15)), wherein the antibody comprisesa heavy chain, wherein the amino acid sequence of the VH chain regioncan comprise any amino acid sequence described herein (e.g., SEQ ID NO:3 or 5), and wherein the constant region of the heavy chain comprisesthe amino acid sequence of a human gamma (γ) heavy chain constantregion. For example, human constant region sequences can be any one ofthose described in U.S. Pat. No. 5,693,780 or Kabat et al. (1971) Ann.NY Acad. Sci. 190:382-391 and, Kabat et al. (1991) Sequences of Proteinsof Immunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242. In particular embodiments,the constant region of the heavy chain comprises the amino acid sequenceof SEQ ID NO: 13.

In yet another specific embodiment, a polynucleotide provided hereincomprises a nucleotide sequence encoding an antibody described herein(or an antigen-binding fragment thereof), which immunospecifically bindsto a KIT polypeptide (e.g., a D4/D5 region of KIT, for example humanKIT), wherein the antibody comprises a VL chain region and a VH chainregion comprising any amino acid sequences described herein, and whereinthe constant regions comprise the amino acid sequences of the constantregions of a human IgG1 (e.g., isotype a, z, or f) or human IgG4. In aparticular embodiment, a polynucleotide provided herein comprises anucleotide sequence encoding an antibody described herein, whichimmunospecifically binds to a KIT polypeptide (e.g., a D4/D5 region ofKIT, for example human KIT), wherein the antibody comprises a VL chainregion and a VH chain region comprising any amino acid sequencesdescribed herein, and wherein the constant regions comprise the aminoacid sequences of the constant region of a human IgG1 (isotype f). Incertain embodiments, the constant region of the light chain comprisesthe amino acid sequence of SEQ ID NO: 12. In particular embodiments, theconstant region of the heavy chain comprises the amino acid sequence ofSEQ ID NO: 13.

In another particular embodiment, a polynucleotide provided hereincomprises a nucleotide sequence encoding an antibody described herein(or an antigen-binding fragment thereof), which immunospecifically bindsto a KIT polypeptide (e.g., a D4/D5 region of KIT, for example humanKIT), wherein the antibody comprises a light chain and a heavy chain,and wherein (i) the light chain comprises a VL chain region comprising aVL CDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ IDNO: 20, 21, and 22, respectively; (ii) the heavy chain comprises a VHchain region comprising a VH CDR1, VH CDR2, and VH CDR3 having the aminoacid sequences of SEQ ID NO: 23, 24, and 25, respectively; (iii) thelight chain further comprises a constant light chain domain comprisingthe amino acid sequence of the constant domain of a human kappa lightchain; and (iv) the heavy chain further comprises a constant heavy chaindomain comprising the amino acid sequence of the constant domain of ahuman IgG1 (optionally IgG1 (isotype f)) heavy chain. In certainembodiments, the constant region of the light chain comprises the aminoacid sequence of SEQ ID NO: 12. In particular embodiments, the constantregion of the heavy chain comprises the amino acid sequence of SEQ IDNO: 13.

In another particular embodiment, a polynucleotide provided hereincomprises a nucleotide sequence encoding an antibody described herein(or an antigen-binding fragment thereof), which immunospecifically bindsto a KIT polypeptide (e.g., a D4/D5 region of KIT, for example humanKIT), wherein the antibody comprises a light chain and a heavy chain,and wherein (i) the light chain comprises a VL chain region comprisingthe amino acid sequence of SEQ ID NO: 2; (ii) the heavy chain comprisesa VH chain region comprising the amino acid sequence of SEQ ID NO: 3;(iii) the light chain further comprises a constant light chain domaincomprising the amino acid sequence of the constant domain of a humankappa light chain; and (iv) the heavy chain further comprises a constantheavy chain domain comprising the amino acid sequence of the constantdomain of a human IgG1 (optionally IgG1 (isotype f)) heavy chain. Incertain embodiments, the constant region of the light chain comprisesthe amino acid sequence of SEQ ID NO: 12. In particular embodiments, theconstant region of the heavy chain comprises the amino acid sequence ofSEQ ID NO: 13.

In another particular embodiment, a polynucleotide provided hereincomprises a nucleotide sequence encoding an antibody described herein,which immunospecifically binds to a KIT polypeptide (e.g., a D4/D5region of KIT, for example human KIT), wherein the antibody comprises alight chain and a heavy chain, and wherein (i) the light chain comprisesa VL chain region comprising the amino acid sequence of SEQ ID NO: 2;(ii) the heavy chain comprises a VH chain region comprising the aminoacid sequence of SEQ ID NO: 5; (iii) the light chain further comprises aconstant light chain domain comprising the amino acid sequence of theconstant domain of a human kappa light chain; and (iv) the heavy chainfurther comprises a constant heavy chain domain comprising the aminoacid sequence of the constant domain of a human IgG1 (optionally IgG1(isotype 0) heavy chain. In certain embodiments, the constant region ofthe light chain comprises the amino acid sequence of SEQ ID NO: 12. Inparticular embodiments, the constant region of the heavy chain comprisesthe amino acid sequence of SEQ ID NO: 13.

In certain embodiments, with respect to a polynucleotide provided hereincomprising a nucleotide sequence encoding a VL chain region and VH chainregion of any of these antibodies described herein, the polynucleotideof the VL chain region further comprises human framework regions; andthe VH chain region further comprises human framework regions.

In certain embodiments, a polynucleotide provided herein comprises anucleotide sequence encoding an antibody described herein, whichimmunospecifically binds to a KIT polypeptide (e.g., a D4/D5 region ofKIT, for example human KIT), wherein the antibody comprises a lightchain and a heavy chain, and wherein (i) the light chain comprises a VLchain region comprising human framework regions; (ii) the heavy chaincomprises a VH chain region comprising human framework regions; (iii)the light chain further comprises a constant light chain domaincomprising the amino acid sequence of the constant domain of a humankappa light chain; and (iv) the heavy chain further comprises a constantheavy chain domain comprising the amino acid sequence of the constantdomain of a human IgG1 (optionally IgG1 (isotype 0) heavy chain. Incertain embodiments, the constant region of the light chain comprisesthe amino acid sequence of SEQ ID NO: 12. In particular embodiments, theconstant region of the heavy chain comprises the amino acid sequence ofSEQ ID NO: 13.

In a specific embodiment, provided herein are polynucleotides comprisinga nucleotide sequence encoding an anti-KIT antibody, or a fragment ordomain thereof, designated herein (see, e.g., Tables 1-4 and FIGS.3A-5C) as antibody 37M or 37C. In a specific embodiment, provided hereinare polynucleotides comprising a nucleotide sequence encoding ananti-KIT antibody, or a fragment or domain thereof, designated herein(see, e.g., Tables 1-4 and FIGS. 3A-5C) as antibody 37C. In a specificembodiment, provided herein are polynucleotides comprising a nucleotidesequence encoding an anti-KIT antibody, or a fragment or domain thereof,designated herein (see, e.g., Tables 1-4 and FIGS. 3A-5C) as antibody37M.

In certain embodiments, polynucleotides described herein comprise anucleotide sequence encoding a light chain or a VL chain regioncomprising the VL CDR1, VL CDR2, and VL CDR3 amino acid sequences ofthose of antibody 37M or 37C (e.g., as designated in Tables 1 and 2). Incertain embodiments, polynucleotides described herein comprise anucleotide sequence encoding a heavy chain or a VH chain regioncomprising the VH CDR1, VH CDR2, and VH CDR3 amino acid sequences ofthose of antibody 37M or 37C (e.g., as designated in Tables 3 and 4). Ina specific embodiment, polynucleotides described herein comprisenucleotide sequences encoding (i) a VL chain region comprising the VLCDR1, VL CDR2, and VL CDR3 amino acid sequences of those of antibody 37Mor 37C (e.g., as designated in Tables 1 and 2); and (ii) a VH chainregion comprising the VH CDR1, VH CDR2, and VH CDR3 amino acid sequencesof those of antibody 37M or 37C (e.g., as designated in Tables 3 and 4).

In certain embodiments, polynucleotides described herein comprise anucleotide sequence encoding a light chain or a VL chain regioncomprising the VL FR1, VL FR2, VL FR3, and VL FR4 amino acid sequencesof those of antibody 37M or 37C (e.g., as designated in Tables 1 and 2).In certain embodiments, polynucleotides described herein comprise anucleotide sequence encoding a heavy chain or a VH chain regioncomprising the VH FR1, VH FR2, VH FR3, and VH FR4 amino acid sequencesof those of antibody 37M or 37C (e.g., as designated in Tables 3 and 4).In a particular embodiment, polynucleotides described herein comprisenucleotide sequences encoding (i) a light chain or a VL chain regioncomprising the VL FR1, VL FR2, VL FR3, and VL FR4 amino acid sequencesof those of antibody 37M or 37C (e.g., as designated in Tables 1 and 2);and (ii) a heavy chain or a VH chain region comprising the VH FR1, VHFR2, VH FR3, and VH FR4 amino acid sequences of those of antibody 37M or37C (e.g., as designated in Tables 3 and 4).

Also provided herein are polynucleotides encoding an anti-KIT antibodyor a fragment thereof that are optimized, e.g., by codon/RNAoptimization, replacement with heterologous signal sequences, andelimination of mRNA instability elements. Methods to generate optimizednucleic acids encoding an anti-KIT antibody or a fragment thereof (e.g.,light chain, heavy chain, VH domain, or VL domain) for recombinantexpression by introducing codon changes and/or eliminating inhibitoryregions in the mRNA can be carried out by adapting the optimizationmethods described in, e.g., U.S. Pat. Nos. 5,965,726; 6,174,666;6,291,664; 6,414,132; and 6,794,498, accordingly. For example, potentialsplice sites and instability elements (e.g., A/T or A/U rich elements)within the RNA can be mutated without altering the amino acids encodedby the nucleic acid sequences to increase stability of the RNA forrecombinant expression. The alterations utilize the degeneracy of thegenetic code, e.g., using an alternative codon for an identical aminoacid. In some embodiments, it can be desirable to alter one or morecodons to encode a conservative mutation, e.g., a similar amino acidwith similar chemical structure and properties and/or function as theoriginal amino acid. Such methods can increase expression of an anti-KITantibody or fragment thereof by at least 1 fold, 2 fold, 3 fold, 4 fold,5 fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold,80 fold, 90 fold, or 100 fold or more relative to the expression of ananti-KIT antibody encoded by polynucleotides that have not beenoptimized.

In certain embodiments, an optimized polynucleotide sequence encoding ananti-KIT antibody described herein or a fragment thereof (e.g., VLdomain and/or VH domain) can hybridize to an antisense polynucleotide ofan unoptimized polynucleotide sequence encoding an anti-KIT antibodydescribed herein or a fragment thereof (e.g., VL domain and/or VHdomain). In specific embodiments, an optimized nucleotide sequenceencoding an anti-KIT antibody described herein or a fragment hybridizesunder high stringency conditions to antisense polynucleotide of anunoptimized polynucleotide sequence encoding an anti-KIT antibodydescribed herein or a fragment thereof. In a specific embodiment, anoptimized nucleotide sequence encoding an anti-KIT antibody describedherein or a fragment thereof hybridizes under high stringency,intermediate or lower stringency hybridization conditions to anantisense polynucleotide of an unoptimized nucleotide sequence encodingan anti-KIT antibody described herein or a fragment thereof. Informationregarding hybridization conditions have been described, see, e.g., U.S.Patent Application Publication No. US 2005/0048549 (e.g., paragraphs72-73), which is incorporated herein by reference in its entirety.

In certain embodiments, an optimized polynucleotide sequence encoding aVL region of an antibody described herein is at least 75%, at least 80%,at least 85%, at least 90%, at least 95%, at least 98% identical to thenucleotide sequence of SEQ ID NO: 8. In certain embodiments, anoptimized polynucleotide sequence encoding a VH region of an antibodydescribed herein is at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98% identical to the nucleotide sequence ofSEQ ID NO: 9.

The polynucleotides can be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. Nucleotidesequences encoding antibodies described herein, e.g., antibodiesdescribed in Tables 1-4, and modified versions of these antibodies canbe determined using methods well known in the art, i.e., nucleotidecodons known to encode particular amino acids are assembled in such away to generate a nucleic acid that encodes the antibody. Such apolynucleotide encoding the antibody can be assembled from chemicallysynthesized oligonucleotides (e.g., as described in Kutmeier et al.,1994, BioTechniques 17:242), which, briefly, involves the synthesis ofoverlapping oligonucleotides containing portions of the sequenceencoding the antibody, annealing and ligating of those oligonucleotides,and then amplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody described hereincan be generated from nucleic acid from a suitable source (e.g., ahybridoma) using methods well known in the art (e.g., PCR and othermolecular cloning methods). For example, PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of a known sequencecan be performed using genomic DNA obtained from hybridoma cellsproducing the antibody of interest. Such PCR amplification methods canbe used to obtain nucleic acids comprising the sequence encoding thelight chain and/or heavy chain of an antibody. Such PCR amplificationmethods can be used to obtain nucleic acids comprising the sequenceencoding the variable light chain region and/or the variable heavy chainregion of an antibody. The amplified nucleic acids can be cloned intovectors for expression in host cells and for further cloning, forexample, to generate chimeric and humanized antibodies.

If a clone containing a nucleic acid encoding a particular antibody isnot available, but the sequence of the antibody molecule is known, anucleic acid encoding the immunoglobulin can be chemically synthesizedor obtained from a suitable source (e.g., an antibody cDNA library or acDNA library generated from, or nucleic acid, preferably poly A+RNA,isolated from, any tissue or cells expressing the antibody, such ashybridoma cells selected to express an antibody described herein) by PCRamplification using synthetic primers hybridizable to the 3′ and 5′ endsof the sequence or by cloning using an oligonucleotide probe specificfor the particular gene sequence to identify, e.g., a cDNA clone from acDNA library that encodes the antibody Amplified nucleic acids generatedby PCR can then be cloned into replicable cloning vectors using anymethod well known in the art.

DNA encoding anti-KIT antibodies described herein can be readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of the anti-KIT antibodies).Hybridoma cells can serve as a source of such DNA. Once isolated, theDNA can be placed into expression vectors, which are then transfectedinto host cells such as E. coli cells, simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of anti-KIT antibodiesin the recombinant host cells.

In phage display methods, functional antibody domains are displayed onthe surface of phage particles which carry the polynucleotide sequencesencoding them. In particular, a library of DNA sequences encoding VH andVL domains are generated (e.g., amplified from animal cDNA librariessuch as human cDNA libraries or random libraries are generated bychemical synthesis). The DNA encoding the VH and VL domains arerecombined together with an scFv linker by PCR and cloned into aphagemid vector. The vector is electroporated in E. coli and the E. coliis infected with helper phage. Phage expressing an antigen-bindingdomain that binds to a particular antigen can be selected or identifiedwith antigen, e.g., using labeled antigen or antigen bound or capturedto a solid surface or bead. After phage selection, the antibody codingregions from the phage can be isolated and used to generate wholeantibodies, including human antibodies, or any other desiredantigen-binding fragment, and expressed in any desired host, includingmammalian cells, insect cells, plant cells, yeast, and bacteria, e.g.,as described below. Techniques to recombinantly produced Fab, Fab′ andF(ab′)₂ fragments can also be employed using methods known in the artsuch as those disclosed in PCT Publication No. WO 92/22324; Mullinax etal., 1992, BioTechniques, 12(6):864-869; Sawai et al., 1995, AJRI,34:26-34; and Better et al., 1988, Science, 240:1041-1043.

Antibodies can be isolated from antibody phage libraries generated usingthe techniques described in McCafferty et al., Nature, 348:552-554(1990). Clackson et al., Nature, 352:624-628 (1991). Marks et al., J.Mol. Biol., 222:581-597 (1991) describe the isolation of murine andhuman antibodies, respectively, using phage libraries. Chain shufflingcan be used in the production of high affinity (nM range) humanantibodies (Marks et al., Bio/Technology, 10:779-783 (1992)), as well ascombinatorial infection and in vivo recombination as a strategy forconstructing very large phage libraries (Waterhouse et al., Nuc. Acids.Res., 21:2265-2266 (1993)).

To generate whole antibodies, PCR primers including VH or VL nucleotidesequences, a restriction site, and a flanking sequence to protect therestriction site can be used to amplify the VH or VL sequences in scFvclones. Utilizing cloning techniques known to those of skill in the art,the PCR amplified VH domains can be cloned into vectors expressing aheavy chain constant region, e.g., the human gamma 4 constant region,and the PCR amplified VL domains can be cloned into vectors expressing alight chain constant region, e.g., human kappa or lambda constantregions. In certain embodiments, the vectors for expressing the VH or VLdomains comprise an EF-1α promoter, a secretion signal, a cloning sitefor the variable domain, constant domains, and a selection marker suchas neomycin. The VH and VL domains can also be cloned into one vectorexpressing the necessary constant regions. The heavy chain conversionvectors and light chain conversion vectors are then co-transfected intocell lines to generate stable or transient cell lines that expressfull-length antibodies, e.g., IgG, using techniques known to those ofskill in the art.

In a non-limiting example, the Dyax (Cambridge, Mass.) technologyplatform can be used to convert Fab-phage or Fabs to complete IgGantibodies, such as the Dyax pR rapid reformatting vectors (RR).Briefly, by PCR, a Fab-encoding DNA fragment is inserted into a DyaxpR-RRV between a eukaryotic leader sequence and an IgG heavy chainconstant region cDNA. Antibody expression is driven by the humancytomegalovirus (hCMV). In a second cloning step, bacterial regulatoryelements are replaced by the appropriate eukaryotic sequences (i.e., theIRES (internal ribosome entry site) motif). The expression vector canalso include the SV40 origin of replication. The Dyax pRh1(a,z),pRh1(f), pRh4 and pRm2a are expression vectors allowing expression ofreformatted FAbs as human IgG1 (isotype a,z), human IgG1 (isotype F),human IgG4, and mouse IgG2a, respectively. Expressing vectors can beintroduced into a suitable host cell (e.g., HEK293T cells, CHO cells))for expression and purification.

The DNA also can be modified, for example, by substituting the codingsequence for human heavy and light chain constant domains in place ofthe murine sequences, or by covalently joining to the immunoglobulincoding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide.

Also provided are polynucleotides that hybridize under high stringency,intermediate or lower stringency hybridization conditions topolynucleotides that encode an antibody described herein. In specificembodiments, polynucleotides described herein hybridize under highstringency, intermediate or lower stringency hybridization conditions topolynucleotides encoding a VH chain region (e.g., SEQ ID NO: 3 or 5)and/or VL chain region (e.g., SEQ ID NO: 2) provided herein. In specificembodiments, polynucleotides described herein hybridize under highstringency or intermediate stringency hybridization conditions topolynucleotides which are complements to polynucleotides encoding a VHchain region (e.g., SEQ ID NO: 3 or 5) and/or VL chain region (e.g., aSEQ ID NO: 2) provided herein.

In specific embodiments, polynucleotides described herein hybridizeunder high stringency, intermediate or lower stringency hybridizationconditions to polynucleotides which are complements to a polynucleotidecomprising SEQ ID NO: 8 or SEQ ID NO: 9. In specific embodiments,polynucleotides described herein hybridize under high stringency orintermediate stringency hybridization conditions to polynucleotideswhich are complements to a polynucleotide comprising SEQ ID NO: 8 or SEQID NO: 9.

In specific embodiments, polynucleotides described herein hybridizeunder high stringency, intermediate or lower stringency hybridizationconditions to polynucleotides which are complements to a polynucleotidecomprising SEQ ID NO: 10 or SEQ ID NO: 11. In specific embodiments,polynucleotides described herein hybridize under high stringency orintermediate stringency hybridization conditions to polynucleotideswhich are complements to a polynucleotide comprising SEQ ID NO: 10 orSEQ ID NO: 11.

Hybridization conditions have been described in the art and are known toone of skill in the art. For example, hybridization under stringentconditions can involve hybridization to filter-bound DNA in 6× sodiumchloride/sodium citrate (SSC) at about 45° C. followed by one or morewashes in 0.2×SSC/0.1% SDS at about 50-65° C.; hybridization underhighly stringent conditions can involve hybridization to filter-boundnucleic acid in 6×SSC at about 45° C. followed by one or more washes in0.1×SSC/0.2% SDS at about 68° C. Hybridization under other stringenthybridization conditions are known to those of skill in the art and havebeen described, see, for example, Ausubel, F. M. et al., eds., 1989,Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc. and John Wiley & Sons, Inc., New York at pages6.3.1-6.3.6 and 2.10.3.

In specific embodiments, polynucleotides described herein have at least80%, 85%, 90%, 95%, or 98% sequence identity to a polynucleotidecomprising SEQ ID NO: 8 encoding a VL chain region of an antibodydescribed herein. In specific embodiments, polynucleotides describedherein have at least 80%, 85%, 90%, 95%, or 98% sequence identity to apolynucleotide comprising SEQ ID NO: 9 encoding a VH chain region of anantibody described herein.

In specific embodiments, polynucleotides described herein have at least80%, 85%, 90%, 95%, or 98% sequence identity to a polynucleotidecomprising SEQ ID NO: 10 encoding a light chain of an antibody describedherein. In specific embodiments, polynucleotides described herein haveat least 80%, 85%, 90%, 95%, or 98% sequence identity to apolynucleotide comprising SEQ ID NO: 11 encoding a heavy chain of anantibody described herein.

5.3 Host Cells and Recombinant Expression of Antibodies

In certain aspects, provided herein are host cells recombinantlyexpressing the antibodies described herein (or an antigen-bindingfragment thereof) and related expression vectors. Provided herein arevectors (e.g., expression vectors) comprising polynucleotides comprisingnucleotide sequences encoding anti-KIT antibodies or a fragment forrecombinant expression in host cells, preferably in mammalian cells.Also provided herein are host cells comprising such vectors forrecombinantly expressing anti-KIT antibodies described herein (e.g.,antibody 37M or 37C). In a particular aspect, provided herein aremethods for producing an antibody described herein, comprisingexpressing such antibody from a host cell.

Recombinant expression of an antibody described herein (e.g., afull-length antibody, heavy and/or light chain of an antibody, or asingle chain antibody described herein) that immunospecifically binds toa KIT antigen involves construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule, heavy and/or light chain of an antibody, orfragment thereof (preferably, but not necessarily, containing the heavyand/or light chain variable domain) described herein has been obtained,the vector for the production of the antibody molecule can be producedby recombinant DNA technology using techniques well-known in the art.Thus, methods for preparing a protein by expressing a polynucleotidecontaining an antibody encoding nucleotide sequence are describedherein. Methods which are well known to those skilled in the art can beused to construct expression vectors containing antibody codingsequences and appropriate transcriptional and translational controlsignals. These methods include, for example, in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination.Also provided are replicable vectors comprising a nucleotide sequenceencoding an antibody molecule described herein, a heavy or light chainof an antibody, a heavy or light chain variable domain of an antibody ora fragment thereof, or a heavy or light chain CDR, operably linked to apromoter. Such vectors can, for example, include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g.,International Publication Nos. WO 86/05807 and WO 89/01036; and U.S.Pat. No. 5,122,464) and the variable domain of the antibody can becloned into such a vector for expression of the entire heavy, the entirelight chain, or both the entire heavy and light chains.

An expression vector can be transferred to a cell (e.g., host cell) byconventional techniques and the resulting cells can then be cultured byconventional techniques to produce an antibody described herein or afragment thereof. Thus, provided herein are host cells containing apolynucleotide encoding an antibody described herein or fragmentsthereof, or a heavy or light chain thereof, or fragment thereof, or asingle chain antibody described herein, operably linked to a promoterfor expression of such sequences in the host cell. In certainembodiments, for the expression of double-chained antibodies, vectorsencoding both the heavy and light chains, individually, can beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below. In certain embodiments, ahost cell contains a vector comprising a polynucleotide encoding boththe heavy chain and light chain of an antibody described herein, or afragment thereof. In specific embodiments, a host cell contains twodifferent vectors, a first vector comprising a polynucleotide encoding aheavy chain of an antibody described herein, or a fragment thereof, anda second vector comprising a polynucleotide encoding a light chain of anantibody described herein, or a fragment thereof. In other embodiments,a first host cell comprises a first vector comprising a polynucleotideencoding a heavy chain of an antibody described herein, or a fragmentthereof, and a second host cell comprises a second vector comprising apolynucleotide encoding a light chain of an antibody described herein.

A variety of host-expression vector systems can be utilized to expressantibody molecules described herein (see, e.g., U.S. Pat. No.5,807,715). Such host-expression systems represent vehicles by which thecoding sequences of interest can be produced and subsequently purified,but also represent cells which can, when transformed or transfected withthe appropriate nucleotide coding sequences, express an antibodymolecule described herein in situ. These include but are not limited tomicroorganisms such as bacteria (e.g., E. coli and B. subtilis)transformed with recombinant bacteriophage DNA, plasmid DNA or cosmidDNA expression vectors containing antibody coding sequences; yeast(e.g., Saccharomyces Pichia) transformed with recombinant yeastexpression vectors containing antibody coding sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing antibody coding sequences; plant cell systems(e.g., green algae such as Chlamydomonas reinhardtii) infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing antibody codingsequences; or mammalian cell systems (e.g., COS, CHO, BHK, MDCK, HEK293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH 3T3 cells)harboring recombinant expression constructs containing promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter). In a specific embodiment, a mammalian expressionvector is pOptiVEC™ or pcDNA3.3. Preferably, bacterial cells such asEscherichia coli, and more preferably, eukaryotic cells, especially forthe expression of whole recombinant antibody molecule, are used for theexpression of a recombinant antibody molecule. For example, mammaliancells such as Chinese hamster ovary (CHO) cells, in conjunction with avector such as the major intermediate early gene promoter element fromhuman cytomegalovirus is an effective expression system for antibodies(Foecking et al., 1986, Gene 45:101; and Cockett et al., 1990,Bio/Technology 8:2). In certain embodiments, antibodies described hereinare produced by CHO cells or NS0 cells. In a specific embodiment, theexpression of nucleotide sequences encoding antibodies described hereinwhich immunospecifically bind to a KIT antigen is regulated by aconstitutive promoter, inducible promoter or tissue specific promoter.

In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such anantibody is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified can be desirable. Such vectors include, but are not limited to,the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO12:1791), in which the antibody coding sequence can be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985,Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol.Chem. 24:5503-5509); and the like. pGEX vectors can also be used toexpress foreign polypeptides as fusion proteins with glutathione5-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence can be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest can be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene can then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts (e.g., see Logan &Shenk, 1984, Proc. Natl. Acad. Sci. USA 8 1:355-359). Specificinitiation signals can also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression canbe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see, e.g., Bittner et al.,1987, Methods in Enzymol. 153:51-544).

In addition, a host cell strain can be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products canbe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product can be used. Such mammalian hostcells include but are not limited to CHO, VERO, BHK, Hela, COS, MDCK,HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (amurine myeloma cell line that does not endogenously produce anyimmunoglobulin chains), CRL7O3O and HsS78Bst cells. In certainembodiments, humanized monoclonal anti-KIT antibodies described hereinare produced in mammalian cells, such as CHO cells.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule can be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells can beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method canadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines can be particularly useful inscreening and evaluation of compositions that interact directly orindirectly with the antibody molecule.

A number of selection systems can be used, including but not limited to,the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell11:223), hypoxanthineguanine phosphoribosyltransferase (Szybalska &Szybalski, 1992, Proc. Natl. Acad. Sci. USA 48:202), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:8-17) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc.Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418 (Wuand Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol.Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan andAnderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH11(5):155-2 15); and hygro, which confers resistance to hygromycin(Santerre et al., 1984, Gene 30:147). Methods commonly known in the artof recombinant DNA technology can be routinely applied to select thedesired recombinant clone, and such methods are described, for example,in Ausubel et al. (eds.), Current Protocols in Molecular Biology, JohnWiley & Sons, N Y (1993); Kriegler, Gene Transfer and Expression, ALaboratory Manual, Stockton Press, N Y (1990); and in Chapters 12 and13, Dracopoli et al. (eds.), Current Protocols in Human Genetics, JohnWiley & Sons, N Y (1994); Colberre-Garapin et al., 1981, J. Mol. Biol.150:1, which are incorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol.3:257).

The host cell can be co-transfected with two or more expression vectorsdescribed herein, the first vector encoding a heavy chain derivedpolypeptide and the second vector encoding a light chain derivedpolypeptide. The two vectors can contain identical selectable markerswhich enable equal expression of heavy and light chain polypeptides. Thehost cells can be co-transfected with different amounts of the two ormore expression vectors. For example, host cells can be transfected withany one of the following ratios of a first expression vector and asecond expression vector: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50.

Alternatively, a single vector can be used which encodes, and is capableof expressing, both heavy and light chain polypeptides. In suchsituations, the light chain should be placed before the heavy chain toavoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature322:52; and Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197-2199). Thecoding sequences for the heavy and light chains can comprise cDNA orgenomic DNA. The expression vector can be monocistronic ormulticistronic. A multicistronic nucleic acid construct can encode 2, 3,4, 5, 6, 7, 8, 9, 10 or more, or in the range of 2-5, 5-10 or 10-20genes/nucleotide sequences. For example, a bicistronic nucleic acidconstruct can comprise in the following order a promoter, a first gene(e.g., heavy chain of an antibody described herein), and a second geneand (e.g., light chain of an antibody described herein). In such anexpression vector, the transcription of both genes can be driven by thepromoter, whereas the translation of the mRNA from the first gene can beby a cap-dependent scanning mechanism and the translation of the mRNAfrom the second gene can be by a cap-independent mechanism, e.g., by anIRES.

Once an antibody molecule described herein has been produced byrecombinant expression, it can be purified by any method known in theart for purification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Further, theantibodies described herein can be fused to heterologous polypeptidesequences described herein or otherwise known in the art to facilitatepurification.

In specific embodiments, an antibody described herein is isolated orpurified. Generally, an isolated antibody is one that is substantiallyfree of other antibodies with different antigenic specificities than theisolated antibody. For example, in a particular embodiment, apreparation of an antibody described herein is substantially free ofcellular material and/or chemical precursors. The language“substantially free of cellular material” includes preparations of anantibody in which the antibody is separated from cellular components ofthe cells from which it is isolated or recombinantly produced. Thus, anantibody that is substantially free of cellular material includespreparations of antibody having less than about 30%, 20%, 10%, 5%, 2%,1%, 0.5%, or 0.1% (by dry weight) of heterologous protein (also referredto herein as a “contaminating protein”) and/or variants of an antibody,for example, different post-translational modified forms of an antibodyor other different versions of an antibody (e.g., antibody fragments).When the antibody is recombinantly produced, it is also generallysubstantially free of culture medium, i.e., culture medium representsless than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of theprotein preparation. When the antibody is produced by chemicalsynthesis, it is generally substantially free of chemical precursors orother chemicals, i.e., it is separated from chemical precursors or otherchemicals which are involved in the synthesis of the protein.Accordingly, such preparations of the antibody have less than about 30%,20%, 10%, or 5% (by dry weight) of chemical precursors or compoundsother than the antibody of interest. In a specific embodiment,antibodies described herein are isolated or purified. 5.4 Methods ofProducing Antibodies

Antibodies described herein (or an antigen-binding fragment thereof)that immunospecifically bind to a KIT antigen can be produced by anymethod known in the art for the synthesis of antibodies, for example, bychemical synthesis or by recombinant expression techniques. The methodsdescribed herein employs, unless otherwise indicated, conventionaltechniques in molecular biology, microbiology, genetic analysis,recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotidesynthesis and modification, nucleic acid hybridization, and relatedfields within the skill of the art. These techniques are described inthe references cited herein and are fully explained in the literature.See, e.g., Maniatis et al. (1982) Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press; Sambrook et al. (1989),Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press; Sambrook et al. (2001) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; Ausubel et al., Current Protocols in Molecular Biology,John Wiley & Sons (1987 and annual updates); Current Protocols inImmunology, John Wiley & Sons (1987 and annual updates) Gait (ed.)(1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press;Eckstein (ed.) (1991) Oligonucleotides and Analogues: A PracticalApproach, IRL Press; Birren et al. (eds.) (1999) Genome Analysis: ALaboratory Manual, Cold Spring Harbor Laboratory Press.

In some embodiments, human antibodies are produced. In particularembodiments, an antibody described herein, which binds to the sameepitope of KIT (e.g., a D4/D5 region of human KIT) as antibody 37M or37C, is a human antibody. In particular embodiments, an antibodydescribed herein, which competitively blocks (e.g., in a dose-dependentmanner) antibody 37M or 37C from binding to KIT (e.g., a D4/D5 region ofhuman KIT), is a human antibody. Human antibodies can be produced usingany method known in the art. For example, transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes, can be used. In particular, thehuman heavy and light chain immunoglobulin gene complexes can beintroduced randomly or by homologous recombination into mouse embryonicstem cells. Alternatively, the human variable region, constant region,and diversity region can be introduced into mouse embryonic stem cellsin addition to the human heavy and light chain genes. The mouse heavyand light chain immunoglobulin genes can be rendered non-functionalseparately or simultaneously with the introduction of humanimmunoglobulin loci by homologous recombination. In particular,homozygous deletion of the J_(H) region prevents endogenous antibodyproduction. The modified embryonic stem cells are expanded andmicroinjected into blastocysts to produce chimeric mice. The chimericmice are then bred to produce homozygous offspring which express humanantibodies. The transgenic mice are immunized in the normal fashion witha selected antigen, e.g., all or a portion of an antigen (e.g., KIT).Monoclonal antibodies directed against the antigen can be obtained fromthe immunized, transgenic mice using conventional hybridoma technology.The human immunoglobulin transgenes harbored by the transgenic micerearrange during B cell differentiation, and subsequently undergo classswitching and somatic mutation. Thus, using such a technique, it ispossible to produce therapeutically useful IgG, IgA, IgM and IgEantibodies. For an overview of this technology for producing humanantibodies, see Lonberg and Huszar, 1995, Int. Rev. Immunol. 13:65-93.For a detailed discussion of this technology for producing humanantibodies and human monoclonal antibodies and protocols for producingsuch antibodies, see, e.g., PCT publication Nos. WO 98/24893, WO96/34096, and WO 96/33735; and U.S. Pat. Nos. 5,413,923, 5,625,126,5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, and 5,939,598.

In some embodiments, human antibodies can be produced using mouse-humanhybridomas. For example, human peripheral blood lymphocytes transformedwith Epstein-Ban virus (EBV) can be fused with mouse myeloma cells toproduce mouse-human hybridomas secreting human monoclonal antibodies,and these mouse-human hybridomas can be screened to determine ones whichsecrete human monoclonal antibodies that immunospecifically bind to atarget antigen (e.g., D4/D5 region of human KIT). Such methods are knownand are described in the art, see, e.g., Shinmoto et al.,Cytotechnology, 2004, 46:19-23; Naganawa et al., Human Antibodies, 2005,14:27-31.

In some embodiments, human antibodies can be generated by insertingpolynucleotides encoding human CDRs (e.g., VL CDRs and/or VH CDRs) of anantibody into an expression vector containing nucleotide sequencesencoding human framework region sequences. In certain embodiments, suchexpression vectors further comprise nucleotide sequences encoding aconstant region of a human light and/or heavy chain. In someembodiments, human antibodies can be generated by inserting human CDRs(e.g., VL CDRs and/or VH CDRs) of an antibody obtained from a phagelibrary into such human expression vectors.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling et al., in: Monoclonal Antibodies and T-CellHybridomas 563 681 (Elsevier, N.Y., 1981). The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. For example,in the hybridoma method, a mouse or other appropriate host animal, suchas a sheep, goat, rabbit, rat, hamster or macaque monkey, is immunizedto elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the protein (e.g., D4/D5region of human KIT) used for immunization. Alternatively, lymphocytesmay be immunized in vitro. Lymphocytes then are fused with myeloma cellsusing a suitable fusing agent, such as polyethylene glycol, to form ahybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice,pp. 59-103 (Academic Press, 1986)). Additionally, a RIMMS (repetitiveimmunization multiple sites) technique can be used to immunize an animal(Kilptrack et al., 1997 Hybridoma 16:381-9, incorporated by reference inits entirety).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Specific embodiments employ myeloma cells that fuse efficiently, supportstable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these myeloma cell lines are murine myeloma lines, such asthose derived from MOPC-21 and MPC-11 mouse tumors available from theSalk Institute Cell Distribution Center, San Diego, Calif., USA, andSP-2 or X63-Ag8.653 cells available from the American Type CultureCollection, Rockville, Md., USA. Human myeloma and mouse-humanheteromyeloma cell lines also have been described for the production ofhuman monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984);Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the human KITantigen. The binding specificity of monoclonal antibodies produced byhybridoma cells is determined by methods known in the art, for example,immunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI 1640 medium. In addition, thehybridoma cells may be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

In some embodiments, mice (or other animals, such as rats, monkeys,donkeys, pigs, sheep, hamster, or dogs) can be immunized with an antigen(e.g., KIT, preferably the D4/D5 region of KIT) and once an immuneresponse is detected, e.g., antibodies specific for the antigen aredetected in the mouse serum, the mouse spleen is harvested andsplenocytes isolated. The splenocytes are then fused by well knowntechniques to any suitable myeloma cells, for example cells from cellline SP20 available from the American Type Culture Collection (ATCC®)(Manassas, Va.), to form hybridomas. Hybridomas are selected and clonedby limited dilution. In certain embodiments, lymph nodes of theimmunized mice are harvested and fused with NS0 myeloma cells.

The hybridoma clones are then assayed by methods known in the art forcells that secrete antibodies capable of binding a polypeptide of theantigen (e.g., human KIT, for example, the D4/D5 region of human KIT).Ascites fluid, which generally contains high levels of antibodies, canbe generated by immunizing mice with positive hybridoma clones.

Accordingly, described herein are methods of generating antibodies byculturing a hybridoma cell secreting an anti-KIT antibody wherein. Inspecific embodiments, the hybridoma is generated by fusing splenocytesisolated from a mouse (or other animal, such as rat, monkey, donkey,pig, sheep, or dog) immunized with a KIT antigen with myeloma cells andthen screening the hybridomas resulting from the fusion for hybridomaclones that secrete an antibody able to bind to the KIT antigen. Incertain embodiments, the hybridoma is generated by fusing lymph nodesisolated from a mouse (or other animal, such as rat, monkey, donkey,pig, sheep, or dog) immunized with a KIT antigen with myeloma cells, andthen screening the hybridomas resulting from the fusion for hybridomaclones that secrete an antibody able to bind to the KIT antigen.

Antibodies described herein include antibody fragments which recognizespecific KIT antigens and can be generated by any technique known tothose of skill in the art. For example, Fab and F(ab′)₂ fragmentsdescribed herein can be produced by proteolytic cleavage ofimmunoglobulin molecules, using enzymes such as papain (to produce Fabfragments) or pepsin (to produce F(ab′)₂ fragments). A Fab fragmentcorresponds to one of the two identical arms of an antibody molecule andcontains the complete light chain paired with the VH and CH1 domains ofthe heavy chain. A F(ab′)₂ fragment contains the two antigen-bindingarms of an antibody molecule linked by disulfide bonds in the hingeregion.

Further, the antibodies described herein can also be generated usingvarious phage display methods known in the art. In phage displaymethods, functional antibody domains are displayed on the surface ofphage particles which carry the polynucleotide sequences encoding them.In particular, DNA sequences encoding VH and VL domains are amplifiedfrom animal cDNA libraries (e.g., human or murine cDNA libraries ofaffected tissues). The DNA encoding the VH and VL domains are recombinedtogether with an scFv linker by PCR and cloned into a phagemid vector.The vector is electroporated in E. coli and the E. coli is infected withhelper phage. Phage used in these methods are typically filamentousphage including fd and M13, and the VH and VL domains are usuallyrecombinantly fused to either the phage gene III or gene VIII. Phageexpressing an antigen binding domain that binds to a particular antigencan be selected or identified with antigen, e.g., using labeled antigenor antigen bound or captured to a solid surface or bead. Examples ofphage display methods that can be used to make the antibodies describedherein include those disclosed in Brinkman et al., 1995, J. Immunol.Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods 184:177-186;Kettleborough et al., 1994, Eur. J. Immunol. 24:952-958; Persic et al.,1997, Gene 187:9-18; Burton et al., 1994, Advances in Immunology57:191-280; PCT Application No. PCT/GB91/O1 134; InternationalPublication Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO93/1 1236, WO 95/15982, WO 95/20401, and WO97/13844; and U.S. Pat. Nos.5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753,5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727,5,733,743 and 5,969,108.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described below. Techniques to recombinantly produceantibody fragments such as Fab, Fab′ and F(ab′)₂ fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication No. WO 92/22324; Mullinax et al., 1992, BioTechniques12(6):864-869; Sawai et al., 1995, AJRI 34:26-34; and Better et al.,1988, Science 240:1041-1043.

In one aspect, to generate whole antibodies, PCR primers including VH orVL nucleotide sequences, a restriction site, and a flanking sequence toprotect the restriction site can be used to amplify the VH or VLsequences from a template, e.g., scFv clones. Utilizing cloningtechniques known to those of skill in the art, the PCR amplified VHdomains can be cloned into vectors expressing a VH constant region, andthe PCR amplified VL domains can be cloned into vectors expressing a VLconstant region, e.g., human kappa or lambda constant regions. The VHand VL domains can also be cloned into one vector expressing thenecessary constant regions. The heavy chain conversion vectors and lightchain conversion vectors are then co-transfected into cell lines togenerate stable or transient cell lines that express full-lengthantibodies, e.g., IgG, using techniques known to those of skill in theart.

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it can be preferable to use human, humanized orchimeric antibodies. Completely human antibodies are particularlydesirable for therapeutic treatment of human subjects. Human antibodiescan be made by a variety of methods known in the art including phagedisplay methods described above using antibody libraries derived fromhuman immunoglobulin sequences. See also U.S. Pat. Nos. 4,444,887 and4,716,111; and International Publication Nos. WO 98/46645, WO 98/50433,WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741.

A chimeric antibody is a molecule in which different portions of theantibody are derived from different immunoglobulin molecules. Forexample, a chimeric antibody can contain a variable region of a mousemonoclonal antibody fused to a constant region of a human antibody.Methods for producing chimeric antibodies are known in the art. See,e.g., Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques4:214; Gillies et al., 1989, J. Immunol. Methods 125:191-202; and U.S.Pat. Nos. 5,807,715, 4,816,567, 4,816,397, and 6,331,415.

A humanized antibody is capable of binding to a predetermined antigenand which comprises a framework region having substantially the aminoacid sequence of a human immunoglobulin and CDRs having substantiallythe amino acid sequence of a non-human immunoglobulin (e.g., a murineimmunoglobulin). In particular embodiments, a humanized antibody alsocomprises at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. The antibody also can includethe CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. Thehumanized antibody can be selected from any class of immunoglobulins,including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG₁,IgG₂, IgG₃ and IgG₄. Humanized antibodies can be produced using avariety of techniques known in the art, including but not limited to,CDR-grafting (European Patent No. EP 239,400; International publicationNo. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and5,585,089), veneering or resurfacing (European Patent Nos. EP 592,106and EP 519,596; Padlan, 1991, Molecular Immunology 28(4/5):489-498;Studnicka et al., 1994, Protein Engineering 7(6):805-814; and Roguska etal., 1994, PNAS 91:969-973), chain shuffling (U.S. Pat. No. 5,565,332),and techniques disclosed in, e.g., U.S. Pat. Nos. 6,407,213, 5,766,886,WO 9317105, Tan et al., J. Immunol. 169:1119 25 (2002), Caldas et al.,Protein Eng. 13(5):353-60 (2000), Morea et al., Methods 20(3):267 79(2000), Baca et al., J. Biol. Chem. 272(16):10678-84 (1997), Roguska etal., Protein Eng. 9(10):895 904 (1996), Couto et al., Cancer Res. 55 (23Supp):5973s-5977s (1995), Couto et al., Cancer Res. 55(8):1717-22(1995), Sandhu J S, Gene 150(2):409-10 (1994), and Pedersen et al., J.Mol. Biol. 235(3):959-73 (1994). See also U.S. Patent Pub. No. US2005/0042664 A1 (Feb. 24, 2005), which is incorporated by referenceherein in its entirety.

Single domain antibodies, for example, antibodies lacking the lightchains, can be produced by methods well-known in the art. See Riechmannet al., 1999, J. Immunol. 231:25-38; Nuttall et al., 2000, Curr. Pharm.Biotechnol. 1(3):253-263; Muylderman, 2001, J. Biotechnol. 74(4):277302;U.S. Pat. No. 6,005,079; and International Publication Nos. WO 94/04678,WO 94/25591, and WO 01/44301.

Further, antibodies that immunospecifically bind to a KIT antigen can,in turn, be utilized to generate anti-idiotype antibodies that “mimic”an antigen using techniques well known to those skilled in the art.(See, e.g., Greenspan & Bona, 1989, FASEB J. 7(5):437-444; andNissinoff, 1991, J. Immunol. 147(8):2429-2438).

5.5 Pharmaceutical Compositions and Kits

Provided herein are compositions, pharmaceutical compositions, and kitscomprising one or more antibodies described herein (or antigen-bindingfragments thereof). In particular aspects, compositions described hereincan be for in vitro, in vivo, or ex vivo uses. In specific embodiments,provided herein is a pharmaceutical composition comprising an antibodydescribed herein (or an antigen-binding fragment thereof) and apharmaceutically acceptable carrier or excipient.

Therapeutic formulations containing one or more antibodies providedherein can be prepared for storage by mixing the antibody having thedesired degree of purity with optional physiologically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences(1990) Mack Publishing Co., Easton, Pa.; Remington: The Science andPractice of Pharmacy, 21st ed. (2006) Lippincott Williams & Wilkins,Baltimore, Md.), in the form of lyophilized formulations or aqueoussolutions. Acceptable carriers, excipients, or stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids; and/ornon-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol(PEG).

Formulations, such as those described herein, can also contain more thanone active compounds (for example, molecules, e.g., antibody orantibodies described herein) as necessary for the particular indicationbeing treated. In certain embodiments, formulations comprise an antibodyprovided herein and one or more active compounds with complementaryactivities that do not adversely affect each other. Such molecules aresuitably present in combination in amounts that are effective for thepurpose intended. For example, an antibody described herein can becombined with one or more other therapeutic agents (e.g., a tyrosinekinase inhibitor such as imatinib mesylated or sunitinib, or a histonedeacetylase inhibitor such as vorinostat). Such combination therapy canbe administered to the patient serially or simultaneously or insequence.

The formulations to be used for in vivo administration can be sterile.This is readily accomplished by filtration through, e.g., sterilefiltration membranes.

In specific aspects, the pharmaceutical compositions provided hereincontain therapeutically effective amounts of one or more of theantibodies provided herein, and optionally one or more additionalprophylactic of therapeutic agents, in a pharmaceutically acceptablecarrier. Such pharmaceutical compositions are useful in the prevention,treatment, management or amelioration of a KIT-mediated disorder ordisease, such as cancer (e.g., GIST) or an inflammatory bowl disease, orone or more of the symptoms thereof.

Pharmaceutical carriers suitable for administration of the antibodiesprovided herein include any such carriers known to those skilled in theart to be suitable for the particular mode of administration.

In addition, the antibodies described herein can be formulated as thesole pharmaceutically active ingredient in the composition or can becombined with other active ingredients (such as one or more otherprophylactic or therapeutic agents).

The compositions can contain one or more antibodies provided herein. Inone embodiment, the antibodies are formulated into suitablepharmaceutical preparations, such as solutions, suspensions, tablets,dispersible tablets, pills, capsules, powders, sustained releaseformulations or elixirs, for oral administration or in sterile solutionsor suspensions for parenteral administration, as well as transdermalpatch preparation and dry powder inhalers.

In the compositions, one or more antibodies provided herein (orconjugates thereof) is (are) mixed with a suitable pharmaceuticalcarrier. The concentrations of the antibody or antibodies in thecompositions can, for example, be effective for delivery of an amount,upon administration, that treats, prevents, or ameliorates aKIT-mediated disorder or disease or a symptom thereof. In particularembodiments, concentrations of an antibody-drug conjugate orantibody-drug conjugates in the compositions can, for example, beeffective for delivery of an amount of a drug(s), upon administration,that treats, prevents, or ameliorates a KIT-mediated disorder or diseaseor a symptom thereof.

In one embodiment, the compositions are formulated for single dosageadministration. To formulate a composition, the weight fraction ofcompound is dissolved, suspended, dispersed or otherwise mixed in aselected carrier at an effective concentration such that the treatedcondition is relieved, prevented, or one or more symptoms areameliorated.

In certain aspects, an antibody provided herein (or an antibody-drugconjugate thereof) is included in the pharmaceutically acceptablecarrier in an effective amount sufficient to exert a therapeuticallyuseful effect in the absence of, or with minimal or negligible,undesirable side effects on the patient treated. A therapeuticallyeffective concentration can be determined empirically by testing thecompounds in in vitro and in vivo systems using routine methods and thenextrapolated therefrom for dosages for humans.

The concentration of antibody in the pharmaceutical composition willdepend on, e.g., the physicochemical characteristics of the antibody,the dosage schedule, and amount administered as well as other factorsknown to those of skill in the art. In certain aspects, theconcentration of antibody-drug conjugate in the pharmaceuticalcomposition will depend on, e.g., the physicochemical characteristics ofthe antibody and/or the drug, the dosage schedule, and amountadministered as well as other factors known to those of skill in theart.

In one embodiment, a therapeutically effective dosage produces a serumconcentration of antibody of from about 0.1 ng/ml to about 50-100 μg/ml.The pharmaceutical compositions, in another embodiment, provide a dosageof from about 0.001 mg to about 2000 mg of antibody per kilogram of bodyweight for administration over a period of time, e.g., every day, everyweek, every 2 weeks, or every 3 weeks. Pharmaceutical dosage unit formscan be prepared to provide from about 0.01 mg to about 2000 mg, and inone embodiment from about 10 mg to about 500 mg of the antibody and/or acombination of other optional essential ingredients per dosage unitform.

In a particular embodiment, an antibody-drug conjugate described hereinis administered at an effective dosage of about 1 to 100 mg ofantibody-drug conjugate per kilogram of body weight for administrationover a period of time, e.g., every day, every week, every 2 weeks, orevery 3 weeks.

The antibody can be administered at once, or can be divided into anumber of smaller doses to be administered at intervals of time. It isunderstood that the precise dosage and duration of treatment is afunction of the disease being treated and can be determined empiricallyusing known testing protocols or by extrapolation from in vivo or invitro test data. It is to be noted that concentrations and dosage valuescan also vary with the severity of the condition to be alleviated. It isto be further understood that for any particular subject, specificdosage regimens can be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions, and that theconcentration ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed compositions.

Upon mixing or addition of the antibody, the resulting mixture can be asolution, suspension, emulsion or the like. The form of the resultingmixture depends upon a number of factors, including the intended mode ofadministration and the solubility of the compound in the selectedcarrier or vehicle. The effective concentration is sufficient forameliorating the symptoms of the disease, disorder or condition treatedand can be empirically determined.

The pharmaceutical compositions are provided for administration tohumans and animals in unit dosage forms, such as sterile parenteralsolutions or suspensions, tablets, capsules, pills, powders, granules,and oral solutions or suspensions, and oil-water emulsions containingsuitable quantities of the compounds or pharmaceutically acceptablederivatives thereof. The antibody is, in one embodiment, formulated andadministered in unit-dosage forms or multiple-dosage forms. Unit-doseforms as used herein refers to physically discrete units suitable forhuman and animal subjects and packaged individually as is known in theart. Each unit-dose contains a predetermined quantity of the antibodysufficient to produce the desired therapeutic effect, in associationwith the required pharmaceutical carrier, vehicle or diluent. Examplesof unit-dose forms include ampoules and syringes and individuallypackaged tablets or capsules. Unit-dose forms can be administered infractions or multiples thereof. A multiple-dose form is a plurality ofidentical unit-dosage forms packaged in a single container to beadministered in segregated unit-dose form. Examples of multiple-doseforms include vials, bottles of tablets or capsules or bottles of pintsor gallons. Hence, multiple dose form is a multiple of unit-doses whichare not segregated in packaging.

In certain embodiments, one or more anti-KIT antibodies described hereinare in a liquid pharmaceutical formulation. Liquid pharmaceuticallyadministrable compositions can, for example, be prepared by dissolving,dispersing, or otherwise mixing an active compound as defined above andoptional pharmaceutical adjuvants in a carrier, such as, for example,water, saline, aqueous dextrose, glycerol, glycols, ethanol, and thelike, to thereby form a solution or suspension. If desired, thepharmaceutical composition to be administered can also contain minoramounts of nontoxic auxiliary substances such as wetting agents,emulsifying agents, solubilizing agents, and pH buffering agents and thelike.

Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in this art; for example, see, e.g.,Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton,Pa.; Remington: The Science and Practice of Pharmacy, 21st ed. (2006)Lippincott Williams & Wilkins, Baltimore, Md.

Dosage forms or compositions containing antibody in the range of 0.005%to 100% with the balance made up from non-toxic carrier can be prepared.Methods for preparation of these compositions are known to those skilledin the art.

Parenteral administration, in one embodiment, is characterized byinjection, either subcutaneously, intramuscularly or intravenously isalso contemplated herein. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution or suspension in liquid prior to injection, or asemulsions. The injectables, solutions and emulsions also contain one ormore excipients. Suitable excipients are, for example, water, saline,dextrose, glycerol or ethanol. In addition, if desired, thepharmaceutical compositions to be administered can also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, stabilizers, solubility enhancers, andother such agents. Other routes of administration may include, entericadministration, intracerebral administration, nasal administration,intraarterial administration, intracardiac administration, intraosseousinfusion, intrathecal administration, and intraperitonealadministration.

Preparations for parenteral administration include sterile solutionsready for injection, sterile dry soluble products, such as lyophilizedpowders, ready to be combined with a solvent just prior to use,including hypodermic tablets, sterile suspensions ready for injection,sterile dry insoluble products ready to be combined with a vehicle justprior to use and sterile emulsions. The solutions can be either aqueousor nonaqueous.

If administered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, and polypropylene glycol and mixtures thereof.

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances.

Pharmaceutical carriers also include ethyl alcohol, polyethylene glycoland propylene glycol for water miscible vehicles; and sodium hydroxide,hydrochloric acid, citric acid or lactic acid for pH adjustment.

Illustratively, intravenous or intraarterial infusion of a sterileaqueous solution containing an active compound is an effective mode ofadministration. Another embodiment is a sterile aqueous or oily solutionor suspension containing an active material injected as necessary toproduce the desired pharmacological effect.

The antibody can be suspended in micronized or other suitable form. Theform of the resulting mixture depends upon a number of factors,including the intended mode of administration and the solubility of thecompound in the selected carrier or vehicle. The effective concentrationis sufficient for ameliorating the symptoms of the condition and can beempirically determined.

In other embodiments, the pharmaceutical formulations are lyophilizedpowders, which can be reconstituted for administration as solutions,emulsions and other mixtures. They can also be reconstituted andformulated as solids or gels.

The lyophilized powder is prepared by dissolving a antibody providedherein, in a suitable solvent. In some embodiments, the lyophilizedpowder is sterile. The solvent can contain an excipient which improvesthe stability or other pharmacological component of the powder orreconstituted solution, prepared from the powder. Excipients that can beused include, but are not limited to, dextrose, sorbital, fructose, cornsyrup, xylitol, glycerin, glucose, sucrose or other suitable agent. Thesolvent can also contain a buffer, such as citrate, sodium or potassiumphosphate or other such buffer known to those of skill in the art at, inone embodiment, about neutral pH. Subsequent sterile filtration of thesolution followed by lyophilization under standard conditions known tothose of skill in the art provides the desired formulation. In oneembodiment, the resulting solution will be apportioned into vials forlyophilization. Each vial will contain a single dosage or multipledosages of the compound. The lyophilized powder can be stored underappropriate conditions, such as at about 4° C. to room temperature.

Reconstitution of this lyophilized powder with water for injectionprovides a formulation for use in parenteral administration. Forreconstitution, the lyophilized powder is added to sterile water orother suitable carrier. The precise amount depends upon the selectedcompound. Such amount can be empirically determined.

Antibodies provided herein can be formulated for local or topicalapplication, such as for topical application to the skin and mucousmembranes, such as in the eye, in the form of gels, creams, and lotionsand for application to the eye or for intracisternal or intraspinalapplication. Topical administration is contemplated for transdermaldelivery and also for administration to the eyes or mucosa, or forinhalation therapies. Nasal solutions of the active compound alone or incombination with other pharmaceutically acceptable excipients can alsobe administered.

The antibodies and other compositions provided herein can also beformulated to be targeted to a particular tissue, receptor, or otherarea of the body of the subject to be treated. Many such targetingmethods are well known to those of skill in the art. All such targetingmethods are contemplated herein for use in the instant compositions. Fornon-limiting examples of targeting methods, see, e.g., U.S. Pat. Nos.6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570,6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534,5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542 and 5,709,874. Insome embodiments, the anti-KIT antibodies described herein are targeted(or otherwise administered) to the bone marrow, such as in a patienthaving or at risk of having leukemia. In some embodiments, anti-KITantibodies described herein are targeted (or otherwise administered) tothe gastrointestinal tract, such as in a patient having or at risk ofhaving gastrointestinal stromal tumors. In some embodiments, anti-KITantibodies described herein are targeted (or otherwise administered) tothe lungs, such as in a patient having or at risk of lung cancer (e.g.,small cell lung cancer). In some embodiments, anti-KIT antibodiesdescribed herein are targeted (or otherwise administered) to the brain,such as in a patient having or at risk of having neuroblastoma. Inspecific embodiments, an anti-KIT antibody described herein is capableof crossing the blood-brain barrier.

Provided herein is a pharmaceutical pack or kit comprising one or morecontainers filled with one or more of the ingredients of thepharmaceutical compositions described herein, such as one or moreantibodies provided herein. Optionally associated with such container(s)can be a notice in the form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals or biologicalproducts, which notice reflects approval by the agency of manufacture,use or sale for human administration.

Also provided herein are kits that can be used in the above methods. Inone embodiment, a kit comprises an antibody described herein, preferablya purified antibody, in one or more containers. In a specificembodiment, the kits described herein contain a substantially isolatedKIT antigen as a control. In another specific embodiment, the kitsdescribed herein further comprise a control antibody which does notreact with the KIT antigen. In another specific embodiment, the kitsdescribed herein contain one or more elements for detecting the bindingof a modified antibody to a KIT antigen (e.g., the antibody can beconjugated to a detectable substrate such as a fluorescent compound, anenzymatic substrate, a radioactive compound or a luminescent compound,or a second antibody which recognizes the first antibody can beconjugated to a detectable substrate). In specific embodiments, the kitcan include a recombinantly produced or chemically synthesized KITantigen. The KIT antigen provided in the kit can also be attached to asolid support. In a more specific embodiment the detecting means of theabove described kit includes a solid support to which KIT antigen isattached. Such a kit can also include a non-attached reporter-labeledanti-human antibody. In this embodiment, binding of the antibody to theKIT antigen can be detected by binding of the said reporter-labeledantibody.

5.6 Therapeutic Methods

Provided herein are methods for impeding, preventing, treating and/ormanaging a KIT-mediated disorder or disease (e.g., cancer). Such methodscomprise administering to a subject in need thereof a therapeuticallyeffective amount of an anti-KIT antibody described herein (e.g.,antibodies 37M and 37C and humanized versions thereof, andantigen-binding fragments thereof). In certain aspects, also providedherein are methods for preventing, treating or managing one or moresymptoms of a KIT-mediated disorder or disease.

In specific embodiments, methods described herein for treating aKIT-mediated disorder or disease provide for the reduction oramelioration of the progression, severity, and/or duration of aKIT-mediated disorder or disease (e.g., cancer, inflammatory condition,or fibrosis) resulting from the administration of one or more therapies(including, but not limited to, the administration of one or moreprophylactic or therapeutic agents, such as an anti-KIT antibodydescribed herein). In further specific embodiments, methods describedherein for treating a KIT-mediated disorder or disease relate toreducing one or more symptoms of a KIT-mediated disorder or disease. Inspecific embodiments, an antibody described herein, such as antibody 37Mor 37C or a humanized version thereof, or an antigen-binding fragmentthereof, or a conjugate thereof, is for use in treating or managing aKIT-mediated disorder (e.g., cancer). In a particular embodiment,provided herein is an antibody for use in treating or managing aKIT-mediated disorder (e.g., cancer), wherein the antibody comprises (i)a VL chain region having the amino acid sequence of SEQ ID NO: 2, and/or(ii) a VH chain region having the amino acid sequence of SEQ ID NO: 3 or5. In another particular embodiment, provided herein is an antibody, oran antigen-binding fragment thereof, for use in treating or managing aKIT-mediated disorder (e.g., cancer), wherein the antibody comprises (i)a VL chain region comprising a VL CDR1, VL CDR2, and VL CDR3, whereinthe VL CDR1 has the amino acid sequence of SEQ ID NO: 20, the VL CDR2has the amino acid sequence of SEQ ID NO: 21, and the VL CDR3 has theamino acid sequence of SEQ ID NO: 22, respectively; and/or (ii) a VHchain region comprising a VH CDR1, VH CDR2, and VH CDR3 having the aminoacid sequences of SEQ ID NO: 23, 24, and 25, respectively: In a specificembodiment, the antibody used in the methods described herein isinternalized by the cell to which it binds. In a particular embodiment,a conjugate is used in the methods described herein, wherein theconjugate comprises an antibody described herein (e.g., antibody 37M or37C), or a KIT-binding fragment thereof. In a specific embodiment, theconjugate comprises an antibody described herein (e.g., antibody 37M or37C), or a KIT-binding fragment thereof, linked, covalently ornon-covalently, to a therapeutic agent, such as a toxin. In a certainembodiment, the conjugate used in the methods described herein isinternalized into a cell to which it binds.

As used herein and unless otherwise specified, the terms “KIT-mediateddisorder” or “KIT-mediated disease” are used interchangeably and referto any disease that is completely or partially caused by, or is theresult of, KIT expression and/or activity or lack thereof. In oneaspect, a KIT-mediated disorder or disease can be known to one of skillin the art or can be ascertained by one of skill in the art. In acertain embodiment, a KIT-mediated disease or disorder is associatedwith KIT expression and/or activity. For example, KIT expression and/oractivity may contribute, in combination with one or more other factors(e.g., mutation or expression and/or activity of another gene), todevelopment and/or progression of a KIT-mediated disease or disorder. Ina certain embodiment, a KIT-mediated disease or disorder is associatedwith one or more mutations of KIT.

In certain embodiments, KIT is aberrantly (e.g., highly) expressed bycells (e.g., on the surface of cells). In particular embodiments, KITexpression is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or 100% higher than KIT expression on the surface of a control cell(e.g., a cell expressing normal levels of KIT, for example, a mast cell,stem cell, brain cell, melanoblast, or ovary cell). In particularembodiments, KIT expression is at least about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, or 100% higher than the average KIT expression onthe surface of a control cell population (e.g., a cell populationexpressing normal levels of KIT, for example, a mast cell population,stem cell population, brain cell population, melanoblast population, orovary cell population). In specific embodiments, such control cells canbe obtained or derived from a healthy individual (e.g., healthy human).In some embodiments, KIT can be aberrantly upregulated in a particularcell type, whether or not KIT is aberrantly expressed on the cellsurface. In particular embodiments, KIT signaling or activity can beaberrantly upregulated in a particular cell type, whether or not KIT isaberrantly expressed on the cell surface. In particular embodiments, KITsignaling is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,or 100% higher than KIT signaling of a control cell (e.g., a cellcontaining normal KIT signaling, for example, a mast cell, stem cell,brain cell, melanoblast, or ovary cell). In particular embodiments, KITsignaling is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,or 100% higher than average KIT signaling of a control cell population(e.g., a cell population containing normal KIT signaling, for example, amast cell population, stem cell population, brain cell population,melanoblast population, or ovary cell population). In certainembodiments, normal, aberrant or excessive cell signaling is caused bybinding of KIT to a KIT ligand. In other embodiments, aberrant orexcessive cell signaling occurs independent of binding of KIT to a KITligand.

In certain embodiments, a KIT-mediated disease is fibrosis or aninflammatory disorder, e.g., inflammatory bowel disease (IBD), such asCrohn's disease (CD) or ulcerative colitis (UC). In other embodiments, aKIT-mediated disease is cancer, such as lung cancer (e.g., small celllung cancer), leukemia, neuroblastoma, melanoma, sarcoma (e.g., Ewing'ssarcoma) or gastrointestinal stromal tumor (GIST).

In certain aspects, a KIT-mediated disorder or disease can becharacterized by gain-of-function KIT activity, increase in KITactivity, or overexpression of KIT. In one embodiment, a KIT-mediateddisorder or disease is completely or partially caused by or is theresult of gain-of-function KIT activity or expression, e.g.,overexpression, of KIT. In certain embodiments, the gain-of-function KITactivity can occur independent of KIT ligand (e.g., SCF) binding KITreceptor. In particular aspects, high or overexpression of KIT in a cellrefers to an expression level which is at least about 35%, 45%, 55%, or65% more than the expression level of a reference cell known to havenormal KIT expression or KIT activity or more than the averageexpression level of KIT in a population of cells or samples known tohave normal KIT expression or KIT activity. Expression levels of KIT canbe assessed by methods described herein or known to one of skill in theart (e.g., Western blotting or immunohistorychemistry). In particularembodiments, a KIT-mediated disorder or disease is characterized by KITactivity which is higher than normal KIT activity and contributes tocellular transformation, neoplasia, and tumorogenesis. In particularaspects, high or increase of KIT activity in a cell refers to a KITactivity level which is at least about 35%, 45%, 55%, or 65% more thanthe expression level of a reference cell known to have normal KITactivity or more than the average level of KIT activity in a populationof cells or samples known to have normal KIT activity. Non-limitingexamples of a KIT activity includes tyrosine phosphorylation of thecytoplasmic domain of KIT, and signaling downstream of KIT, such as Stator Akt signaling.

Non-limiting examples of disorders or KIT-mediated disorders or diseasesinclude cancers such as breast cancer, leukemia (e.g., chronicmyelogenous leukemia, acute myeloid leukemia, mast cell leukemia), lungcancer (e.g., small cell lung cancer), neuroblastoma, gastrointestinalstromal tumors (GIST), melanoma, colorectal cancer, sarcoma (e.g.,Ewing's sarcoma), and germ cell tumors (e.g., seminoma). In a particularembodiment, a cancer which is treated or managed by the methods providedherein is characterized by a gain-of-function KIT mutation oroverexpression of KIT.

In a specific embodiment, a method described herein is for treatingcancer (e.g., GIST, lung cancer, or sarcoma (e.g., Ewing's sarcoma)),wherein said method comprises administering to a subject in need thereofa therapeutically effective amount of an antibody described herein(e.g., antibody 37M or 37C, or an antigen binding fragment thereof(e.g., KIT-binding fragment thereof, for example 3 VL domain CDRs and/or3 VH domain CDRs), or a humanized version of antibody 37M or 37C, or aconjugate comprising, for example an antibody comprising CDRs ofantibody 37M or 37C (or a KIT-binding fragment thereof) linked,covalently or non-covalently, to a therapeutic agent). In certainaspects, also provided herein are methods for preventing, treating ormanaging one or more symptoms of cancer, wherein said methods compriseadministering to a subject in need thereof a therapeutically effectiveamount of an antibody described herein (e.g., antibody 37M or 37C, or anantigen binding fragment thereof (e.g., KIT-binding fragment thereof),or a humanized version of antibody 37M or 37C, or a conjugatecomprising, for example an antibody comprising CDRs of antibody 37M or37C or a KIT-binding fragment thereof, linked, covalently ornon-covalently, to a therapeutic agent). In a specific embodiment, anantibody for use in the methods of treating cancer described hereincomprises a VL domain comprising the amino acid sequence of SEQ ID NO: 2(or CDRs of SEQ ID NO: 2), and/or a VH domain comprising the amino acidsequence of SEQ ID NO: 3 or 5 (or CDRs of SEQ ID NO: 3 or 5). In aspecific embodiment, an antibody for use in the methods of treatingcancer described herein comprises CDRs of antibody 37M or 37C (e.g., VLCDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NO:20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; and/or VH CDR1, VHCDR2, and VH CDR3 having the amino acid sequences of SEQ ID NO: 23, SEQID NO: 24, and SEQ ID NO: 25, respectively).

In a specific embodiment, a method described herein is for treatingGIST, wherein said method comprises administering to a subject in needthereof a therapeutically effective amount of an antibody describedherein (e.g., antibody 37M or 37C, or an antigen binding fragmentthereof (e.g., KIT-binding fragment thereof), or a humanized version ofantibody 37M or 37C, or a conjugate comprising, for example an antibodycomprising CDRs of antibody 37M or 37C or a KIT-binding fragmentthereof, linked, covalently or non-covalently, to a therapeutic agent).In certain aspects, also provided herein are methods for preventing,treating or managing one or more symptoms of GIST, wherein said methodscomprise administering to a subject in need thereof a therapeuticallyeffective amount of an antibody described herein (e.g., antibody 37M or37C, or an antigen binding fragment thereof (e.g., KIT-binding fragmentthereof), or a humanized version of antibody 37M or 37C, or a conjugatecomprising, for example an antibody comprising the CDRs of antibody 37Mor 37C or a KIT-binding fragment thereof, linked, covalently ornon-covalently, to a therapeutic agent). In a specific embodiment, anantibody for use in the methods of treating GIST described hereincomprises a VL domain comprising the amino acid sequence of SEQ ID NO: 2(or CDRs of SEQ ID NO: 2), and/or a VH domain comprising the amino acidsequence of SEQ ID NO: 3 or 5 (or CDRs of SEQ ID NO: 3 or 5). In aspecific embodiment, an antibody for use in the methods of treating GISTdescribed herein comprises CDRs of antibody 37M or 37C (e.g., VL CDR1,VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NO: 20,SEQ ID NO: 21, and SEQ ID NO: 22, respectively; and/or VH CDR1, VH CDR2,and VH CDR3 having the amino acid sequences of SEQ ID NO: 23, SEQ ID NO:24, and SEQ ID NO: 25, respectively).

In a specific embodiment, a method described herein is for treating lungcancer (e.g., small cell lung carcinoma), wherein said method comprisesadministering to a subject in need thereof a therapeutically effectiveamount of an antibody described herein (e.g., antibody 37M or 37C, or anantigen binding fragment thereof, or a conjugate comprising for examplean antibody comprising CDRs of antibody 37M or 37C or a KIT-bindingfragment thereof linked, covalently or non-covalently, to a therapeuticagent). In certain aspects, also provided herein are methods forpreventing, treating or managing one or more symptoms of lung cancer(e.g., small cell lung carcinoma), wherein said methods compriseadministering to a subject in need thereof a therapeutically effectiveamount of an antibody described herein (e.g., antibody 37M or 37C, or anantigen binding fragment thereof (e.g., KIT-binding fragment thereof),or a humanized version of antibody 37M or 37C, or a conjugatecomprising, for example an antibody comprising CDRs of antibody 37M or37C, linked, covalently or non-covalently, to a therapeutic agent). In aspecific embodiment, an antibody for use in the methods of treating lungcancer (e.g., small cell lung cancer) comprises a VL domain comprisingthe amino acid sequence of SEQ ID NO: 2 (or CDRs of SEQ ID NO: 2),and/or a VH domain comprising the amino acid sequence of SEQ ID NO: 3 or5 (or CDRs of SEQ ID NO: 3 or 5). In a specific embodiment, an antibodyfor use in the methods for treating lung cancer (e.g., small cell lungcancer) described herein comprises CDRs of antibody 37M or 37C (e.g., VLCDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NO:20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; and/or VH CDR1, VHCDR2, and VH CDR3 having the amino acid sequences of SEQ ID NO: 23, SEQID NO: 24, and SEQ ID NO: 25, respectively).

In a specific embodiment, a method described herein is for treatingmelanoma, wherein said method comprises administering to a subject inneed thereof a therapeutically effective amount of an antibody describedherein (e.g., antibody 37M or 37C, or an antigen binding fragmentthereof, or a humanized antibody of antibody 37M or 37C, or a conjugatecomprising for example an antibody comprising CDRs of antibody 37M or37C, linked, covalently or non-covalently, to a therapeutic agent). Incertain aspects, also provided herein are methods for preventing,treating or managing one or more symptoms of melanoma, wherein saidmethods comprise administering to a subject in need thereof atherapeutically effective amount of an antibody described herein (e.g.,antibody 37M or 37C, or an antigen binding fragment thereof, or ahumanized antibody of antibody 37M or 37C, or a conjugate comprising forexample an antibody comprising CDRs of antibody 37M or 37C, linked,covalently or non-covalently, to a therapeutic agent). In a specificembodiment, an antibody for use in the methods of treating melanomadescribed herein comprises a VL domain comprising the amino acidsequence of SEQ ID NO: 2 (or CDRs of SEQ ID NO: 2), and/or a VH domaincomprising the amino acid sequence of SEQ ID NO: 3 or 5 (or CDRs of SEQID NO: 3 or 5). In a specific embodiment, an antibody for use in themethods for treating melanoma described herein comprises CDRs ofantibody 37M or 37C (e.g., VL CDR1, VL CDR2, and VL CDR3 having theamino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22,respectively; and/or VH CDR1, VH CDR2, and VH CDR3 having the amino acidsequences of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25,respectively).

In specific embodiments, a cancer treated in accordance with the methodsdescribed herein can be any type of cancer which comprises cancer ortumor cells expressing KIT or a mutated form thereof, which can beconfirmed by any histologically or cytologically method known to one ofskill in the art.

In certain embodiments, a cancer is metastatic. In certain embodiments,a cancer is an advanced cancer which has spread outside the site ororgan of origin, either by local invasion or metastasis.

In particular embodiments, a cancer is a recurrent cancer which hasregrown, either at the initial site or at a distant site, after aresponse to initial therapy (e.g., after surgery to remove the tumor andadjuvant therapy following surgery). In some embodiments, a cancer is arefractory cancer which progresses even though an anti-tumor agent, suchas a chemotherapeutic agent, is being administered, or has beenadministered, to the cancer patient. A non-limiting example of arefractory cancer is one which is refractory to a tyrosine kinaseinhibitor, such as GLEEVEC® (imatinib mesylate), SUTENT® (SU11248 orsunitinib), IRESSA™ (gefitinib), TARCEVA® (erlotinib), NEXAVAR®(sorafenib), or VOTRIENT™ (pazopanib). In some embodiments, a cancer isa refractory cancer which progresses even though radiation orchemotherapy is being administered, or has been administered, to thecancer patient.

In specific embodiments, provided herein are methods for treating arefractory cancer in a patient in need thereof comprising administeringto the patient a therapeutically effective amount of an antibodydescribed herein, wherein the refractory cancer is refractory orresistant to an anti-cancer agent such as a tyrosine kinase inhibitor(e.g., GLEEVEC® (imatinib mesylate) or SUTENT® (SU11248 or Sunitinib)).Other non-limiting examples of tyrosine kinse inhibitors include 706 andAMNI07 (nilotinib). RAD00I, PKC412, gefitinib (IRESSA™), erlotinib(TARCEVA®), sorafenib (NEXAVAR®), pazopanib (VOTRIENT™), axitinib,bosutinib, cediranib (RECENTIN®), SPRYCEL® (dasatinib), lapatinib(TYKERB®), lestaurtinib, neratinib, nilotinib (TASIGNA®), semaxanib,toceranib (PALLADIA™), vandetanib (ZACTIMA™), and vatalanib. In certainembodiments, the refractory cancer was initially responsive to ananti-cancer agent, such as a tyrosine kinase inhibitor (e.g., GLEEVEC®or SU11248 (i.e., sunitinib)), but has developed resistance theanti-cancer agent. In certain embodiments, a subject has one or moremutations in KIT that confers resistance to an anti-cancer agent such asa tyrosine kinase inhibitor.

In particular embodiments, an antibody described herein is administeredto a patient who has previously received, or is currently receiving, oneor more anti-cancer therapies, for example, a chemotherapeutic agent, ora tyrosine kinase inhibitor (e.g., GLEEVEC® (imatinib mesylate), SUTENT®(SU11248 or sunitinib), IRESSA™ (gefitinib), TARCEVA® (erlotinib),NEXAVAR® (sorafenib), or VOTRIENT™ (pazopanib)) or a histone deacetylaseinhibitor (e.g., vorinostat or suberoylanilide hydroxamic acid (SAHA)).In other particular embodiments, an antibody described herein isadministered to a patient who is, or is suspected of being, resistant orrefractory to an anti-cancer therapy, for example, a tyrosine kinaseinhibitor, e.g., GLEEVEC® (imatinib mesylate), SUTENT® (SU11248 orsunitinib), IRESSA™ (gefitinib), TARCEVA® (erlotinib), NEXAVAR®(sorafenib), or VOTRIENT™ (pazopanib).

In particular embodiments, an antibody described herein (e.g., antibody37M or 37C, or an antigen binding fragment thereof (e.g., KIT-bindingfragment thereof), or a humanized version of antibody 37M or 37C, or aconjugate comprising, for example an antibody comprising CDRs ofantibody 37M or 37C, linked, covalently or non-covalently, to atherapeutic agent) is administered to a patient who has previouslyreceived, or is currently receiving, one or more anti-cancer therapies,for example, an anti-growth factor receptor antibody (e.g., anti-HER2antibody, anti-EGFR antibody, anti-VEGFR antibody, or anti-KITantibody), or anti-growth factor antibody (e.g., anti-EGF antibody,anti-VEGF antibody). In other particular embodiments, an antibodydescribed herein is administered to a patient who is, or is suspected ofbeing, resistant or refractory to an anti-cancer therapy, for example,an anti-growth factor receptor antibody (e.g., anti-HER2 antibody,anti-EGFR antibody, anti-VEGFR antibody, or anti-KIT antibody) oranti-growth factor antibody (e.g., anti-EGF antibody, anti-VEGFantibody).

In a particular embodiment, a method described herein for treating ormanaging cancer in a subject in need thereof, can achieve at least one,two, three, four or more of the following effects due to administrationof a therapeutically effective amount of an anti-KIT antibody describedherein: (i) the reduction or amelioration of the severity of cancer(e.g., leukemia, lung cancer, or gastrointestinal stromal cancer) and/orone or more symptoms associated therewith; (ii) the reduction in theduration of one or more symptoms associated with a cancer (e.g.,leukemia, lung cancer, or gastrointestinal stromal cancer); (iii) theprevention in the recurrence of a tumor (e.g., lung tumor orgastrointestinal stromal tumor); (iv) the regression of a cancer (e.g.,leukemia, lung cancer, or gastrointestinal stromal tumor) and/or one ormore symptoms associated therewith; (v) the reduction in hospitalizationof a subject; (vi) the reduction in hospitalization length; (vii) theincrease in the survival of a subject; (viii) the inhibition of theprogression of a cancer (e.g., leukemia, lung cancer, orgastrointestinal stromal tumor) and/or one or more symptoms associatedtherewith; (ix) the enhancement or improvement of the therapeutic effectof another therapy (e.g., surgery, radiation, chemotherapy, or anothertyrosine kinase inhibitor); (x) a reduction or elimination in the cancercell population (e.g., leukemia cell population, lung cancer cellpopulation, gastrointestinal stromal tumor cell population); (xi) areduction in the growth of a tumor or neoplasm; (xii) a decrease intumor size (e.g., volume or diameter); (xiii) a reduction in theformation of a newly formed tumors; (xiv) eradication, removal, orcontrol of primary, regional and/or metastatic cancer; (xv) ease inremoval of a tumor by reducing tumor and/or edema-relatedvascularization prior to surgery; (xvi) a decrease in the number or sizeof metastases; (xvii) a reduction in mortality; (xviii) an increase intumor-free survival rate of patients; (xvix) an increase in relapse-freesurvival; (xx) an increase in the number of patients in remission; (xxi)a decrease in hospitalization rate; (xxii) the size of the tumor ismaintained and does not increase or increases by less than the increaseof a tumor after administration of a standard therapy as measured byconventional methods available to one of skill in the art, such ascomputed tomography (CT) scan, magnetic resonance imaging (MRI), dynamiccontrast-enhanced MRI (DCE-MRI), or a positron emission tomography (PET)scan; (xxiii) the prevention of the development or onset of one or moresymptoms associated cancer; (xxiv) an increase in the length ofremission in patients; (xxv) the reduction in the number of symptomsassociated with cancer; (xxvi) an increase in symptom-free survival ofcancer patients; (xxvii) a decrease in the concentration of one or moreinflammatory mediators (e.g., cytokines or interleukins) in biologicalspecimens (e.g., plasma, serum, cerebral spinal fluid, urine, or anyother biofluids) of a subject with a cancer (e.g., leukemia, lungcancer, or gastrointestinal stromal cancer); (xxviii) a decrease incirculating tumor cells (CTCs) in the blood of a subject with cancer(e.g., leukemia, lung cancer, or gastrointestinal stromal cancer);(xxix) inhibition (e.g., partial inhibition) or decrease in tumormetabolism or perfusion; and (xxx) improvement in the quality of life asassessed by methods well known in the art, e.g., questionnaires.

In certain aspects, provided herein are methods for killing cancer cellsin an individual in need thereof, wherein said method comprisesadministering to said individual an effective amount of an antibodydescribed herein (e.g., antibody 37M or 37C, or an antigen bindingfragment thereof (e.g., KIT-binding fragment thereof), or a humanizedversion of antibody 37M or 37C, or a conjugate comprising, for examplean antibody comprising CDRs of antibody 37M or 37C, linked, covalentlyor non-covalently, to a therapeutic agent). In certain aspects, providedherein are methods for inhibiting growth or proliferation of cancercells in an individual in need thereof, wherein said method comprisesadministering to said individual an effective amount of an antibodydescribed herein (e.g., antibody 37M or 37C, or an antigen bindingfragment thereof (e.g., KIT-binding fragment thereof), or a humanizedversion of antibody 37M or 37C, or a conjugate comprising, for exampleantibody 37M or 37C, a KIT-binding fragment thereof, or an antibodycomprising CDRs of antibody 37M or 37C, linked, covalently ornon-covalently, to a therapeutic agent). In certain embodiments, partialinhibition of growth or proliferation of cancer cells is achieved, forexample, inhibition of at least about 20% to about 55% of growth orproliferation of cancer cells.

In certain aspects, provided herein are methods for reducing tumor sizeor load in an individual in need thereof, wherein said method comprisesadministering to said individual an effective amount of an antibodydescribed herein (e.g., antibody 37M or 37C, or an antigen bindingfragment thereof (e.g., KIT-binding fragment thereof), or a humanizedversion of antibody 37M or 37C, or a conjugate comprising, for examplean antibody comprising CDRs of antibody 37M or 37C, linked, covalentlyor non-covalently, to a therapeutic agent).

Other non-limiting examples of KIT-mediated disorders or diseasesinclude systemic mast cell disorders (e.g., mastocytosis), hematologicdisorders, fibrosis (e.g., idiopathic pulmonary fibrosis (TPF),scleroderma, or myelofibrosis) and inflammatory conditions such asasthma, rheumatoid arthritis, inflammatory bowel disease, and allergicinflammation.

In a particular embodiment, a method described herein for treating ormanaging a KIT-mediated disorder, e.g., fibrosis or an inflammatorycondition (e.g., asthma, rheumatoid arthritis, inflammatory boweldisease, and allergic inflammation), in a subject in need thereof, canachieve at least one, two, three, four or more of the following effectsdue to administration of a therapeutically effective amount of ananti-KIT antibody described herein: (i) the reduction or amelioration ofthe severity of fibrosis or an inflammatory condition (e.g., asthma,rheumatoid arthritis, inflammatory bowel disease, and allergicinflammation) and/or one or more symptoms associated therewith; (ii) thereduction in the duration of one or more symptoms associated withfibrosis or an inflammatory condition (e.g., asthma, rheumatoidarthritis, inflammatory bowel disease, and allergic inflammation); (iii)the prevention in the recurrence of fibrosis or an inflammatorycondition (e.g., asthma, rheumatoid arthritis, inflammatory boweldisease, and allergic inflammation); (iv) the reduction inhospitalization of a subject; (v) the reduction in hospitalizationlength; (vi) the inhibition (e.g., partial inhibition) of theprogression of fibrosis or an inflammatory condition (e.g., asthma,rheumatoid arthritis, inflammatory bowel disease, and allergicinflammation) and/or one or more symptoms associated therewith; (vii)the enhancement or improvement of the therapeutic effect of anothertherapy (e.g., anti-inflammatory therapy such as steroids); (viii) anincrease in the number of patients in remission (i.e., a time periodcharacterized by no or minimal symptoms associated with the inflammatorycondition); (ix) an increase in the length of remission in patients; (x)a decrease in hospitalization rate; (xi) the reduction in the number ofsymptoms associated with fibrosis or an inflammatory condition (e.g.,asthma, rheumatoid arthritis, inflammatory bowel disease, and allergicinflammation); (xii) a decrease in the concentration of one or moreinflammatory mediators (e.g., cytokines or interleukins) in biologicalspecimens (e.g., plasma, serum, cerebral spinal fluid, urine, or anyother biofluids) of a subject with fibrosis or an inflammatory condition(e.g., asthma, rheumatoid arthritis, inflammatory bowel disease, andallergic inflammation); and (xiii) improvement in the quality of life asassessed by methods well known in the art, e.g., questionnaires.

In certain embodiments, an anti-KIT antibody described herein may beadministered by any suitable method to a subject in need thereof.Non-limiting examples of administration methods include mucosal,intradermal, intravenous, intratumoral, subcutaneous, intramusculardelivery and/or any other method of physical delivery described hereinor known in the art. In one embodiment, an anti-KIT antibody or apharmaceutical composition thereof is administered systemically (e.g.,parenterally) to a subject in need thereof. In another embodiment, ananti-KIT antibody or a pharmaceutical composition thereof isadministered locally (e.g., intratumorally) to a subject in needthereof. Each dose may or may not be administered by an identical routeof administration. In some embodiments, an anti-KIT antibody describedherein can be administered via multiple routes of administrationsimultaneously or subsequently to other doses of the same or a differentan anti-KIT antibody described herein.

When a disease, or a symptom thereof, is being treated, administrationof the substance typically occurs after the onset of the disease orsymptoms thereof. When a disease, or symptoms thereof, are beingprevented, administration of the substance typically occurs before theonset of the disease or symptoms thereof. In certain embodiments, ananti-KIT antibody described herein is administered prophylactically ortherapeutically to a subject. An anti-KIT antibody described herein canbe prophylactically or therapeutically administered to a subject so asto prevent, lessen or ameliorate a KIT-mediated disorder or disease(e.g., cancer, inflammatory condition, fibrosis) or symptom thereof.

The dosage and frequency of administration of an anti-KIT antibodydescribed herein or a pharmaceutical composition thereof is administeredto a subject in need thereof in accordance with the methods for treatinga KIT-mediated disorder or disease provided herein will be efficaciouswhile minimizing side effects. The exact dosage of an anti-KIT antibodydescribed herein to be administered to a particular subject or apharmaceutical composition thereof can be determined by a practitioner,in light of factors related to the subject that requires treatment.Factors which can be taken into account include the severity of thedisease state, general health of the subject, age, and weight of thesubject, diet, time and frequency of administration, combination(s) withother therapeutic agents or drugs, reaction sensitivities, andtolerance/response to therapy. The dosage and frequency ofadministration of an anti-KIT antibody described herein or apharmaceutical composition thereof can be adjusted over time to providesufficient levels of the anti-KIT antibody or to maintain the desiredeffect.

The precise dose to be employed in the formulation will also depend onthe route of administration, and the seriousness of a KIT-mediateddisorder or disease (e.g., cancer, inflammatory condition, fibrosis),and should be decided according to the judgment of the practitioner andeach patient's circumstances.

Effective doses can be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

In one embodiment, for the anti-KIT antibodies described herein, thedosage administered to a patient, to manage a KIT-mediated disorder ordisease (e.g., cancer, inflammatory condition, fibrosis) is typically0.1 mg/kg to 100 mg/kg of the patient's body weight. Generally, humanantibodies have a longer half-life within the human body than antibodiesfrom other species due to the immune response to the foreignpolypeptides. Thus, lower dosages of human antibodies and less frequentadministration is often possible. Further, the dosage and frequency ofadministration of the antibodies described herein can be reduced byenhancing uptake and tissue penetration of the antibodies bymodifications such as, for example, lipidation.

In one embodiment, approximately 0.001 mg/kg (mg of antibody per kgweight of a subject) to approximately 500 mg/kg of an anti-KIT antibodydescribed herein is administered to manage a KIT-mediated disorder ordisease (e.g., cancer, inflammatory condition, fibrosis).

In some embodiments, an effective amount of an antibody provided hereinis from about 0.01 mg to about 1,000 mg. In specific embodiments, an“effective amount” of an anti-KIT antibody described herein refers to anamount of an anti-KIT antibody described herein which is sufficient toachieve at least one, two, three, four or more of the following effects:(i) the reduction or amelioration of the severity of a KIT-mediateddisorder or disease (e.g., cancer, inflammatory condition, fibrosis)and/or one or more symptoms associated therewith; (ii) the reduction inthe duration of one or more symptoms associated with a KIT-mediateddisorder or disease (e.g., cancer, inflammatory condition, fibrosis);(iii) the prevention in the recurrence of a tumor (e.g.,gastrointestinal stromal tumor); (iv) the regression of a KIT-mediateddisorder or disease (e.g., cancer, inflammatory condition, fibrosis)and/or one or more symptoms associated therewith; (v) the reduction inhospitalization of a subject; (vi) the reduction in hospitalizationlength; (vii) the increase in the survival of a subject; (viii) theinhibition (e.g., partial inhibition) of the progression of aKIT-mediated disorder or disease (e.g., cancer, inflammatory condition,fibrosis) and/or one or more symptoms associated therewith; (ix) theenhancement or improvement of the therapeutic effect of another therapy;(x) a reduction or elimination in the cancer cell population (e.g.,leukemia cell population, lung cancer cell population, gastrointestinalstromal cancer cell population); (xi) a reduction in the growth of atumor or neoplasm; (xii) a decrease in tumor size (e.g., volume ordiameter); (xiii) a reduction in the formation of a newly formed tumors;(xiv) eradication, removal, or control of primary, regional and/ormetastatic cancer; (xv) ease in removal of a tumor by reducing tumorand/or edema-related vascularization prior to surgery; (xvi) a decreasein the number or size of metastases; (xvii) a reduction in mortality;(xviii) an increase in tumor-free survival rate of patients; (xvix) anincrease in relapse-free survival; (xx) an increase in the number ofpatients in remission; (xxi) a decrease in hospitalization rate; (xxii)the size of the tumor is maintained and does not increase or increasesby less than the increase of a tumor after administration of a standardtherapy as measured by conventional methods available to one of skill inthe art, such as computed tomography (CT) scan, magnetic resonanceimaging (MRI), dynamic contrast-enhanced MRI (DCE-MRI), or a positronemission tomography (PET) scan; (xxiii) the prevention of thedevelopment or onset of one or more symptoms associated cancer; (xxiv)an increase in the length of remission in patients; (xxv) the reductionin the number of symptoms associated with cancer; (xxvi) an increase insymptom-free survival of cancer patients; (xxvii) a decrease in theconcentration of one or more inflammatory mediators (e.g., cytokines orinterleukins) in biological specimens (e.g., plasma, serum, cerebralspinal fluid, urine, or any other biofluids) of a subject with aKIT-mediated disorder or disease (e.g., cancer, inflammatory condition,fibrosis); (xxviii) a decrease in circulating tumor cells (CTCs) in theblood of a subject with cancer; (xxix) inhibition (e.g., partialinhibition) or decrease in tumor metabolism or perfusion; and (xxx)improvement in the quality of life as assessed by methods well known inthe art, e.g., questionnaires. In some embodiments, “effective amount”as used herein also refers to the amount of an antibody described hereinto achieve a specified result (e.g., inhibition of one or more KITbiological activities of a cell, such as inhibition of cellproliferation).

In some embodiments, an anti-KIT antibody described herein isadministered as necessary, e.g., weekly, biweekly (i.e., once every twoweeks), monthly, bimonthly, trimonthly, etc., as determined by aphysician.

In some embodiments, a single dose of an anti-KIT antibody describedherein is administered one or more times to a patient to impede,prevent, manage, treat and/or ameliorate a KIT-mediated disorder ordisease (e.g., cancer, inflammatory condition, fibrosis).

In particular embodiments, an anti-KIT antibody or pharmaceuticalcomposition thereof is administered to a subject in accordance with themethods for treating a KIT-mediated disorder or disease (e.g., cancer,inflammatory condition, fibrosis) presented herein in cycles, whereinthe anti-KIT antibody or pharmaceutical composition is administered fora period of time, followed by a period of rest (i.e., the anti-KITantibody or pharmaceutical composition is not administered for a periodof time).

Also, presented herein are combination therapies for the treatment of aKIT-mediated disorder or disease (e.g., cancer, inflammatory condition,fibrosis) which involve the administration of an anti-KIT antibodydescribed herein (e.g., antibody 37M or 37C, or an antigen bindingfragment thereof (e.g., KIT-binding fragment thereof), or a humanizedversion of antibody 37M or 37C, or a conjugate comprising, for examplean antibody comprising CDRs of antibody 37M or 37C, linked, covalentlyor non-covalently, to a therapeutic agent) in combination with one ormore additional therapies (e.g., chemotherapeutic agent, tyrosine kinaseinhibitor, or histone deacetylase inhibitor) to a subject in needthereof. In a specific embodiment, presented herein are combinationtherapies for the treatment of a KIT-mediated disorder or disease (e.g.,cancer, inflammatory condition, fibrosis) which involve theadministration of an amount (e.g., a therapeutically effective amount ora sub-optimal amount) of an anti-KIT antibody described herein incombination with an amount (e.g., a therapeutically effective amount ora sub-optimal amount) of another therapy (e.g., chemotherapeutic agent,tyrosine kinase inhibitor, or histone deacetylase inhibitor) to asubject in need thereof.

In combination therapies, one or more anti-KIT antibodies providedherein (e.g., antibody 37M or 37C, or an antigen binding fragmentthereof (e.g., KIT-binding fragment thereof), or a humanized version ofantibody 37M or 37C, or a conjugate comprising, for example an antibodycomprising CDRs of antibody 37M or 37C, linked, covalently ornon-covalently, to a therapeutic agent) can be administered prior to,concurrently with, or subsequent to the administration of one or moreadditional therapies (e.g., agents, surgery, or radiation) for use intreating, managing, and/or ameoliorating a KIT-mediated disorder ordisease (e.g., cancer, inflammatory condition, fibrosis). The use of theterm “in combination” does not restrict the order in which one or moreanti-KIT antibodies and one or more additional therapies areadministered to a subject. In specific embodiments, the therapies can beadministered serially or sequentially.

In specific embodiments, one or more anti-KIT antibodies provided herein(e.g., antibody 37M or 37C, or an antigen binding fragment thereof(e.g., KIT-binding fragment thereof), or a humanized version of antibody37M or 37C, or a conjugate comprising, for example an antibodycomprising CDRs of antibody 37M or 37C, linked, covalently ornon-covalently, to a therapeutic agent) can be administered prior to,concurrently with, or subsequent to the administration of one or moreadditional therapies such as anticancer agents, for example, tyrosinekinase inhibitors (e.g., imatinib myselyate (Gleevec®) or sunitinib(SUTENT), or histone deacetylase inhibitors (e.g., vorinostat orsuberoylanilide hydroxamic acid (SAHA)), for treating, managing, and/orameoliorating a KIT-mediated disorder or disease (e.g., cancer, forexample, GIST, melanoma, or lung cancer).

In another specific embodiment, presented herein are combinationtherapies for the treatment of a KIT-mediated disorder or disease (e.g.,cancer, inflammatory condition, fibrosis) which involve theadministration of an amount of an anti-KIT antibody described herein(e.g., antibody 37M or 37C, or an antigen binding fragment thereof(e.g., KIT-binding fragment thereof), or a humanized version of antibody37M or 37C, or a conjugate comprising, for example an antibodycomprising CDRs of antibody 37M or 37C, linked, covalently ornon-covalently, to a therapeutic agent) in combination with an amount ofanother therapy (e.g., chemotherapeutic agent, tyrosine kinaseinhibitor, or histone deacetylase inhibitor) to a subject in needthereof. In a specific embodiment, the combination therapies result in asynergistic effect. In certain embodiments, the combination therapiesresult in an additive effect.

In a specific embodiment, presented herein are combination therapies forthe treatment of cancer which involve the administration of an amount ofan anti-KIT antibody described herein in combination with an amount ofanother therapy (e.g., surgery, radiation, stem cell transplantation, orchemotherapy) to a subject in need thereof. In a specific embodiment,the combination therapies result in a synergistic effect. In anotherspecific embodiment, the combination therapies result in an additiveeffect.

In a specific embodiment, presented herein are combination therapies forthe treatment of an inflammatory condition which involve theadministration of an amount of an anti-KIT antibody described herein incombination with an amount of another therapy (e.g., anti-inflammatorytherapy, for example, steroid therapy) to a subject in need thereof. Ina specific embodiment, the combination therapies result in a synergisticeffect. In another specific embodiment, the combination therapies resultin an additive effect.

Non-limiting examples of another therapy for use in combination withantibodies described herein include, another anti-KIT antibody thatimmunospecifically bind to a different epitope of KIT, one or more otherantibodies (e.g., anti-HER2 antibody, anti-EGFR antibody, anti-VEGFantibody), anti-inflammatory therapy, chemotherapy (e.g., microtubuledisassembly blocker, antimetabolite, topisomerase inhibitor, and DNAcrosslinker or damaging agent), radiation, surgery, and tyrosine kinaseinhibitors (e.g., imatinib mesylate (GLEEVEC®), sunitinib (SUTENT® orSU11248), gefitinib (IRESSA™), erlotinib (TARCEVA®), sorafenib(NEXAVAR®), pazopanib (VOTRIENT™), axitinib, bosutinib, cediranib(RECENTIN®), SPRYCEL® (dasatinib), lapatinib (TYKERB®), lestaurtinib,neratinib, nilotinib (TASIGNA®), semaxanib, toceranib (PALLADIA™),vandetanib (ZACTIMA™), and vatalanib). In a specific embodiment, anothertherapy for use in combination with antibodies described herein isimatinib mesylate.

Other non-limiting examples of another therapy for use in combinationwith antibodies described herein (an antibody comprising CDRs ofantibody 37M or 37C or a conjugate comprising an antibody comprisingCDRs of antibody 37M and an agent) include a histone deacetylaseinhibitor, such as vorinostat or suberoylanilide hydroxamic acid (SAHA)or a compound having the chemical formula (I), (II), or (III) as setforth below. In a specific embodiment, provided herein is a method fortreating cancer (e.g., GIST or lung cancer) comprising (i) administeringan antibody (or antigen-binding fragment thereof) comprising a VL chainregion comprising a VL CDR1, VL CDR2, and VL CDR3 having the amino acidsequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22,respectively; and/or a VH chain region comprising a VH CDR1, VH CDR2,and VH CDR3 having the amino acid sequences of SEQ ID NO: 23, SEQ ID NO:24, and SEQ ID NO: 25, respectively, or an antibody conjugate comprisingsuch antibody and an agent, wherein said antibody, or antigen-bindingfragment thereof, specifically binds to a KIT antigen; and (ii) ahistone deacetylase inhibitor, for example, vorinostat orsuberoylanilide hydroxamic acid (SAHA) or a compound having the chemicalformula (I), (II), or (III) as set forth below. In a specificembodiment, provided herein is a method for treating cancer (e.g., GISTor lung cancer) comprising (i) administering an antibody (or anantigen-binding fragment thereof) comprising a VL chain regioncomprising VL CDR1, VL CDR2, and VL CDR3 of SEQ ID NO: 2; and/or a VHchain region comprising VH CDR1, VH CDR2, and VH CDR3 or SEQ ID NO: 3 or5, or an antibody conjugate comprising such antibody and an agent,wherein said antibody, or antigen-binding fragment thereof, specificallybinds to a KIT antigen; and (ii) a histone deacetylase inhibitor, forexample, vorinostat or suberoylanilide hydroxamic acid (SAHA) or acompound having the chemical formula (I), (II), or (III) as set forthbelow.

In one embodiment, provided herein for use in the methods describedherein in combination with anti-KIT antibodies are compounds of Formula(I)

or a pharmaceutically acceptable salt, hydrate, or solvate thereof,wherein

-   R₁ is hydroxylamino;-   each of R₂ and R₃ are independently the same as or different from    each other, substituted or unsubstituted, branched or unbranched,    and are hydrogen, hydroxyl, alkyl, alkenyl, cycloalkyl, aryl,    alkyloxy, aryloxy, arylalkyloxy or pyridine; or R₂ and R₃ are bonded    together to form a piperidine; and-   n is an integer from 5 to 7.

In one embodiment, R₂ is hydrogen atom and R₃ is substituted orunsubstituted phenyl. In a certain embodiment, R₃ is phenyl substitutedwith methyl, cyano, nitro, trifluoromethyl, amino, aminocarbonyl,methylcyano, chloro, fluoro, bromo, iodo, 2,3-difluoro, 2,4-difluoro,2,5-difluoro, 3,4-difluoro, 3,5-difluoro, 2,6-difluoro, 1,2,3-trifluoro,2,3,6-trifluoro, 2,4,6-trifluoro, 3,4,5-trifluoro, 2,3,5,6-tetrafluoro,2,3,4,5,6-pentafluoro, azido, hexyl, t-butyl, phenyl, carboxyl,hydroxyl, methoxy, phenyloxy, benzyloxy, phenylaminooxy,phenylaminocarbonyl, methoxycarbonyl, methylaminocarbonyl,dimethylamino, dimethylaminocarbonyl, or hydroxylaminocarbonyl. Inanother embodiment, R₃ is unsubstituted phenyl. In a further embodiment,n is 6.

In one embodiment, provided herein for use in the methods describedherein in combination with anti-KIT antibodies are compounds of Formula(II)

or a pharmaceutically acceptable salt, or solvate thereof, wherein n isan integer from 5 to 8. In one embodiment n is 6.

In one embodiment, provided herein for use in the methods describedherein in combination with anti-KIT antibodies is a compound of Formula(III) (SAHA)

or a pharmaceutically acceptable salt, hydrate or solvate thereof.

Compounds of Formulae I-III can be synthesized according to the methodsdescribed in U.S. Reissued Pat. No. RE38,506 and U.S. Pat. No.6,087,367, each of which is herewith incorporated by reference in itsentirety.

In one embodiment, provided herein for use in the methods describedherein in combination with anti-KIT antibodies is a Form I polymorph ofSAHA characterized by an X-ray diffraction pattern substantially similarto that set forth in FIG. 13A of U.S. Pat. No. 7,456,219, which isherewith incorporated by reference in its entirety. In one embodimentthe Form I polymorph of SAHA is characterized by an X-ray diffractionpattern including characteristic peaks at about 9.0, 9.4, 17.5, 19.4,20.0, 24.0, 24.4, 24.8, 25.0, 28.0, and 43.3 degrees 2θ, as measuredwith a Siemens D500 Automated Powder Diffractometer (range: 4-40 degrees2θ; source: Cu; λ=1.54 Angstrom, 50 kV, 40 mA).

In a certain embodiment, the Form I polymorph of SAHA is characterizedby a Differential Scanning calorimetry (DSC) thermogram having a singlemaximum value at about 164.4±2.0° C., as measured by a Perkins Elmer DSC6 Instrument at a heating rate of 10° C./min from 50° C. to at least 30°C. above the observed melting temperature.

The Form I polymorph of SAHA can be synthesized according to the methodsdescribed in U.S. Pat. No. 7,456,219.

In one embodiment, provided herein is a crystalline compositioncomprising Lysine and SAHA characterized by an X-ray diffraction patternsubstantially similar to that set forth in FIG. 1 of InternationalPatent Application Publication No. WO2008/042146, which is herewithincorporated by reference in its entirety. In another embodiment, thecrystalline composition is characterized by an X-ray diffraction patternincluding characteristic peaks at about 6.8, 20.1 and 23.2 degrees 2θ,as measured with a PANanalytical X'Pert Pro X-ray powder diffractometer(range: 2-40 degrees 2θ; source: Cu Kα1 and Kα2). In another embodiment,the crystalline composition is characterized by an X-ray diffractionpattern including characteristic peaks at about 6.8, 12.6, 18.7, 20.123.2, and 24.0 degrees 2θ, as measured with a PANanalytical X'Pert ProX-ray powder diffractometer (range: 2-40 degrees 2θ; source: Cu Kα1 andKα2). In another embodiment, the crystalline composition ischaracterized by an X-ray diffraction pattern including characteristicpeaks at about 6.8, 12.0, 12.6, 16.4, 18.7, 20.1 23.2, 24.0, 29.3degrees 2θ, as measured with a PANanalytical X'Pert Pro X-ray powderdiffractometer (range: 2-40 degrees 2θ; source: Cu Kα1 and Kα2).

In a certain embodiment, the crystalline composition comprising Lysineand SAHA is characterized by a Differential Scanning calorimetry (DSC)thermogram, wherein the endotherm of the crytalline composition exhibitsan extrapolated onset temperature of approximately 182° C., as measuredby a TA Instruments Q1000 differential scanning calorimeter at a heatingrate of 10° C./min from room temperature to 300° C.

The crystalline composition comprising Lysine and SAHA can besynthesized according to the methods described in International PatentApplication Publication No. WO2008/042146.

In certain embodiments, combination therapies described herein result insynergy or a synergistic effect. In a specific embodiment, a synergisticeffect of a combination therapy permits the use of lower dosages (e.g.,sub-optimal doses) of an anti-KIT antibody described herein and/or anadditional therapy and/or less frequent administration of an anti-KITantibody described herein or an additional therapy to a subject. Incertain embodiments, the ability to utilize lower dosages of an anti-KITantibody and/or of an additional therapy and/or to administer ananti-KIT antibody or said additional therapy less frequently reduces thetoxicity associated with the administration of an anti-KIT antibody orof said additional therapy, respectively, to a subject without reducingthe efficacy of an anti-KIT antibody or of said additional therapy,respectively, in the treatment of a KIT-mediated disorder or disease. Insome embodiments, a synergistic effect results in improved efficacy ofan anti-KIT antibody described herein and/or of said additionaltherapies in treating a KIT-mediated disorder or disease. In someembodiments, a synergistic effect of a combination of an anti-KITantibody described herein and one or more additional therapies avoids orreduces adverse or unwanted side effects associated with the use of anysingle therapy.

5.7 Diagnostic Methods

Labeled or otherwise detectable antibodies, which immunospecificallybind to a KIT antigen (e.g., the D4/D5 region of KIT, for example, humanKIT) can be used for diagnostic purposes to detect, diagnose, or monitora KIT-mediated disease.

Provided herein are methods for detecting KIT expression in samplesobtained from patients with a KIT-mediated disorder or disease. In aparticular embodiment, a method for detecting KIT expression in a sampleobtained from a patient comprises contacting the sample with an anti-KITantibody described herein and detecting the expression level of KIT inthe samples. Methods for detection are known to one of skill in the art.

In certain aspects, provided herein are methods for diagnosing a patientwith a KIT-mediated disorder or disease. In a certain aspect, a methodfor diagnosing a subject with a KIT-mediated disorder or diseasecomprises contacting a sample obtained from the subject with an anti-KITantibody described herein (or an antigen-binding fragment thereof) anddetecting the expression level of KIT in the sample. In certainembodiments, a method for diagnosing a patient with a KIT-mediateddisorder or disease is an in vitro method. In particular embodiments, amethod for diagnosing a patient with a KIT-mediated disorder or diseaseis an ex vivo method.

In certain aspects, provided herein are methods for the detection of aKIT-mediated disease comprising: (a) assaying the expression of a KITantigen in cells or a tissue sample of a subject using one or moreantibodies described herein; and (b) comparing the level of the KITantigen with a control level, e.g., levels in normal tissue samples(e.g., from a patient not having a KIT-mediated disease, or from thesame patient before disease onset), whereby an increase in the assayedlevel of KIT antigen compared to the control level of the KIT antigen isindicative of a KIT-mediated disease.

Methods for detection are known to one of skill in the art. For example,the anti-KIT antibody can be conjugated to a detectable molecule (e.g.,as described in section 5.1.1), and the detectable molecule can bevisualized using standard techniques (e.g., microscopy). Antibodiesdescribed herein can be used to assay KIT antigen levels in a biologicalsample using classical immunohistological methods as described herein oras known to those of skill in the art (e.g., see Jalkanen et al., 1985,J. Cell. Biol. 101:976-985; and Jalkanen et al., 1987, J. Cell. Biol.105:3087-3096). Other antibody-based methods useful for detectingprotein gene expression include immunoassays, such as ELISA and theradioimmunoassay (RIA). Suitable antibody assay labels are known in theart and include enzyme labels, such as, glucose oxidase; radioisotopes,such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium (³H),indium (¹²¹In), and technetium (⁹⁹Tc); luminescent labels, such asluminol; and fluorescent labels, such as fluorescein and rhodamine, andbiotin. In specific embodiments, diagnostic methods described hereininvolve using naked or unlabeled antibodies not conjugated to adetectable marker, and the naked or unlabeled antibodies are detectedindirectly, e.g., by using a secondary antibody, which can be labeled.

In certain embodiments, high expression of KIT in a sample relative to anormal control sample (e.g., sample obtained from a healthy patient notsuffering from a KIT-mediated disorder or disease) indicates that thepatient is suffering from a KIT-mediated disorder or disease.

A method for diagnosing a patient with a KIT-mediated disorder ordisease, such as cancer, in a sample obtained from a patient comprisescontacting the sample with an anti-KIT antibody described herein anddetecting the expression level of KIT in the sample. In certainembodiments, high expression of KIT in a sample relative to a normalcontrol sample (e.g., sample obtained from a healthy patient notsuffering from a KIT-mediated disorder or disease) indicates that thepatient is suffering from a KIT-mediated disorder or disease.

In certain embodiments, a sample can be a tumor sample derived from, orcomprising tumor cells from, a patient's tumor. Examples of tumorsamples herein include, but are not limited to, tumor biopsies,circulating tumor cells, circulating plasma proteins, ascitic fluid,primary cell cultures or cell lines derived from tumors or exhibitingtumor-like properties, as well as preserved tumor samples, such asformalin-fixed, paraffin-embedded tumor samples or frozen tumor samples.In certain embodiments, a sample is a fixed tumor sample which has beenhistologically preserved using a fixative. In some embodiments, a sampleis a formalin-fixed tumor sample which has been preserved usingformaldehyde as the fixative. In certain embodiments, a sample is anembedded tumor sample which is surrounded by a firm and generally hardmedium such as paraffin, wax, celloidin, or a resin. Embedding makespossible the cutting of thin sections for microscopic examination or forgeneration of tissue microarrays (TMAs). In particular embodiments, asample is a paraffin-embedded tumor sample which is surrounded by apurified mixture of solid hydrocarbons derived from petroleum. Incertain embodiments, a sample is a frozen tumor sample which is, or hasbeen, frozen. In a specific embodiment, a sample, for example, aparaffin-embedded sample or frozen sample, is sectioned.

In certain aspects, a cancer or biological sample which displays KITexpression, amplification, or activation is one which, in a diagnostictest, expresses (including overexpresses) a KIT receptor, has amplifiedKIT gene, and/or otherwise demonstrates activation or phosphorylation ofa KIT receptor.

Also provided herein is the detection and diagnosis of a KIT-mediateddisease in a human. In one embodiment, diagnosis comprises: a)administering (for example, parenterally, subcutaneously, orintraperitoneally) to a subject an effective amount of a labeledantibody described herein; b) waiting for a time interval following theadministering for permitting the labeled antibody to preferentiallyconcentrate at sites in the subject where the KIT antigen is expressed(and for unbound labeled molecule to be cleared to background level); c)determining background level; and d) detecting the labeled antibody inthe subject, such that detection of labeled antibody above thebackground level indicates that the subject has a KIT-mediated disease.Background level can be determined by various methods including,comparing the amount of labeled molecule detected to a standard valuepreviously determined for a particular system.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of ⁹⁹Tc. The labeled antibody willthen preferentially accumulate at the location of cells which containthe specific protein. In vivo tumor imaging is described in S. W.Burchiel et al., “Immunopharmacokinetics of Radiolabeled Antibodies andTheir Fragments.” (Chapter 13 in Tumor Imaging: The RadiochemicalDetection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., MassonPublishing Inc. (1982).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled antibody to preferentially concentrate atsites in the subject and for unbound labeled antibody to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In one embodiment, monitoring of a KIT-mediated disease is carried outby repeating the method for diagnosing the a KIT-mediated disease, forexample, one month after initial diagnosis, six months after initialdiagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the subject usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat can be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patient using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

5.8 Methods

Provided herein are methods for inhibiting KIT activity in a cellexpressing KIT comprising contacting the cell with an effective amountof an antibody described herein (e.g., antibody 37M or 37C, or anantigen binding fragment thereof (e.g., KIT-binding fragment thereof),or a humanized version of antibody 37M or 37C, or a conjugatecomprising, for example antibody 37M or 37C, a KIT-binding fragmentthereof, or an antibody comprising CDRs of antibody 37M or 37C, linked,covalently or non-covalently, to a therapeutic agent). Also providedherein are methods for inducing or enhancing apoptosis in a cellexpressing KIT comprising contacting the cell with an effective amountof an antibody described herein. Also provided herein are methods forinducing or enhancing cell differentiation in a cell expressing KITcomprising contacting the cell with an effective amount of an antibodydescribed herein.

KIT activity and, for example, the effect of an antibody on KIT activitycan routinely be assessed using, e.g., cell-based assays such as thosedescribed herein.

Non-limiting examples of KIT activity which can be inhibited by themethods provided herein can include any activity of KIT known ordescribed in the art, e.g., KIT receptor dimerization, KIT receptorphosphorylation (tyrosine phosphorylation), signaling downstream of theKIT receptor (e.g., Stat, AKT, MAPK, or Ras signaling), KIT ligand(e.g., SCF) induced transcriptional regulation (e.g., SCF-inducedtranscriptional activation of c-Myc), induction or enhancement of cellproliferation, or cell survival.

In certain embodiments, a method for inhibiting (e.g., partiallyinhibiting) KIT activity in a cell expressing KIT comprises contactingthe cell with an effective amount of an antibody described herein (e.g.,antibody 37M or 37C, or an antigen binding fragment thereof (e.g.,KIT-binding fragment thereof), or a humanized version of antibody 37M or37C, or a conjugate comprising, for example antibody 37M or 37C, aKIT-binding fragment thereof, or an antibody comprising CDRs of antibody37M or 37C, linked, covalently or non-covalently, to a therapeuticagent) sufficient to inhibit or antagonize KIT activity by at leastabout 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 98%, or 99% as assessed by methods describedherein and/or known to one of skill in the art (e.g., ELISA). In certainembodiments, a method for inhibiting (e.g., partially inhibiting) KITactivity in a cell expressing KIT comprises contacting the cell with aneffective amount of an antibody described herein (e.g., antibody 37M or37C, or an antigen binding fragment thereof (e.g., KIT-binding fragmentthereof), or a humanized version of antibody 37M or 37C, or a conjugatecomprising, for example antibody 37M or 37C, a KIT-binding fragmentthereof, or an antibody comprising CDRs of antibody 37M or 37C, linked,covalently or non-covalently, to a therapeutic agent) sufficient toinhibit or antagonize KIT activity by at least about 25%, 35%, 45%, 55%,or 65%, as assessed by methods described herein and/or known to one ofskill in the art (e.g., ELISA). Non-limiting examples of KIT activitycan include KIT receptor phosphorylation, KIT receptor signaling, KITligand (e.g., SCF) mediated cell proliferation, KIT ligand (e.g., SCF)mediated cell survival, and transcriptional activation of a KIT targetgene (e.g., c-Myc).

In a particular embodiment, a method for inhibiting KIT activity in acell expressing KIT comprises contacting the cell with an effectiveamount of an antibody described herein (e.g., antibody 37M or 37C, or anantigen binding fragment thereof (e.g., KIT-binding fragment thereof),or a humanized version of antibody 37M or 37C, or a conjugatecomprising, for example antibody 37M or 37C, a KIT-binding fragmentthereof, or an antibody comprising CDRs of antibody 37M or 37C, linked,covalently or non-covalently, to a therapeutic agent) sufficient toinhibit (e.g., partially inhibit) or antagonize downstream KITsignaling, for example, signaling of a member of the Src family kinases,PI 3-kinases, or Ras-MAPK.

In another particular embodiment, a method for inhibiting (e.g.,partially inhibiting) one or more KIT activities in a cell expressingKIT, comprises contacting the cell with an effective amount of anantibody described herein sufficient to inhibit or antagonize downstreamKIT signaling such as phosphorylation of MAPK, phosphorylation of AKT,or phosphorylation of Stat1, Stat3, or Stat5. Thus, in certainembodiments, a method for an inhibiting (e.g., partially inhibiting) orantagonizing KIT activity in a cell expressing KIT comprises contactingthe cell with an effective amount of an antibody described hereinsufficient to inhibit or to reduce phosphorylation of MAPK (e.g., KITligand (e.g., SCF) induced phosphorylation of MAPK) by at least about5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 98%, or 99% as assessed by methods describedherein or known to one of skill in the art, e.g., Western blot or ELISAassay as described in section 6 or immunoblotting assay. In certainembodiments, a method for an inhibiting (e.g., partially inhibiting) orantagonizing KIT activity in a cell expressing KIT comprises contactingthe cell with an effective amount of an antibody described hereinsufficient to inhibit or to reduce phosphorylation of MAPK (e.g., KITligand (e.g., SCF) induced phosphorylation of MAPK) by at least about25%, 35%, 45%, 55%, or 65%, as assessed by methods described herein orknown to one of skill in the art, e.g., Western blot or ELISA assay asdescribed in section 6 or immunoblotting assay.

In certain embodiments, a method for inhibiting (e.g., partiallyinhibiting) KIT activity in a cell expressing KIT comprises contactingthe cell with an effective amount of an antibody described hereinsufficient to inhibit or to reduce phosphorylation of AKT (e.g., KITligand (e.g., SCF) induced phosphorylation of AKT) by at least about 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 98%, or 99% as assessed by methods described hereinor known to one of skill in the art, e.g., Western blot or ELISA assayas described in section 6 or immunoblotting assay. In certainembodiments, a method for inhibiting e.g., partially inhibiting) KITactivity in a cell expressing KIT comprises contacting the cell with aneffective amount of an antibody described herein sufficient to inhibitor to reduce phosphorylation of AKT (e.g., KIT ligand (e.g., SCF)induced phosphorylation of AKT) by at least about 25%, 35%, 45%, 55%, or65%, as assessed by methods described herein or known to one of skill inthe art, e.g., Western blot or ELISA assay as described in section 6 orimmunoblotting assay.

In particular embodiments, a method for inhibiting (e.g., partiallyinhibiting) KIT activity in a cell expressing KIT comprises contactingthe cell with an effective amount of an antibody described hereinsufficient to inhibit or reduce phosphorylation of Stat3 (e.g., KITligand (e.g., SCF) induced phosphorylation of Stat3) by at least about5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 98%, or 99% as assessed by methods describedherein or known to one of skill in the art, e.g., Western blot or ELISAassay as described in section 6 or immunoblotting assay. In particularembodiments, a method for inhibiting (e.g., partially inhibiting) KITactivity in a cell expressing KIT comprises contacting the cell with aneffective amount of an antibody described herein sufficient to inhibitor reduce phosphorylation of Stat3 (e.g., KIT ligand (e.g., SCF) inducedphosphorylation of Stat3) by at least about 25%, 35%, 45%, 55%, or 65%,as assessed by methods described herein or known to one of skill in theart, e.g., Western blot or ELISA assay as described in section 6 orimmunoblotting assay.

In particular embodiments, a method for inhibiting (e.g., partiallyinhibiting) KIT activity in a cell expressing KIT comprises contactingthe cell with an effective amount of an antibody described hereinsufficient to inhibit or reduce phosphorylation of Stat1 or Stat5 (e.g.,KIT ligand (e.g., SCF) induced phosphorylation) by at least about 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 98%, or 99% as assessed by methods described hereinor known to one of skill in the art, e.g., Western blot or ELISA assayas described in section 6 or immunoblotting assay. In particularembodiments, a method for inhibiting (e.g., partially inhibiting) KITactivity in a cell expressing KIT comprises contacting the cell with aneffective amount of an antibody described herein sufficient to inhibitor reduce phosphorylation of Stat1 or Stat5 (e.g., KIT ligand (e.g.,SCF) induced phosphorylation) by at least about 25%, 35%, 45%, 55%, or65%, as assessed by methods described herein or known to one of skill inthe art, e.g., Western blot or ELISA assay as described in section 6 orimmunoblotting assay.

In certain aspects, a method for inhibiting (e.g., partially inhibiting)KIT activity in a cell expressing KIT comprises contacting the cell withan effective amount of an antibody described herein sufficient toinhibit proliferation of the cell. Cell proliferation assays aredescribed in the art and can be readily carried out by one of skill inthe art. For example, cell proliferation can be assayed by measuringBromodeoxyuridine (BrdU) incorporation (see, e.g., Hoshino et al., 1986,Int. J. Cancer 38, 369; Campana et al., 1988, J. Immunol. Meth. 107:79)or (3H) thymidine incorporation (see, e.g., Blechman et al., Cell, 1995,80:103-113; Chen, J., 1996, Oncogene 13:1395-403; Jeoung, J., 1995, J.Biol. Chem. 270:18367 73), by direct cell count at various timeintervals (e.g., 12-hour or 24-hour intervals), or by detecting changesin transcription, translation or activity of known genes such asproto-oncogenes (e.g., fos, myc) or cell cycle markers (Rb, cdc2, cyclinA, D1, D2, D3, E, etc). The levels of such protein and mRNA and activitycan be determined by any method well known in the art. For example,protein can be quantitated by known immunodiagnostic methods such asELISA, Western blotting or immunoprecipitation using antibodies,including commercially available antibodies. mRNA can be quantitatedusing methods that are well known and routine in the art, for example,using northern analysis, RNase protection, or polymerase chain reactionin connection with reverse transcription.

In specific embodiments, a method for inhibiting (e.g., partiallyinhibiting) KIT activity in cells expressing KIT comprises contactingthe cells with an effective amount of an antibody described hereinsufficient to inhibit cell proliferation by at least about 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 98%, or 99% as assessed by methods described herein or knownto one of skill in the art (e.g., BrdU incorporation assay). In specificembodiments, a method for inhibiting (e.g., partially inhibiting) KITactivity in cells expressing KIT comprises contacting the cells with aneffective amount of an antibody described herein sufficient to inhibitcell proliferation by at least about 25%, 35%, 45%, 55%, or 65%, asassessed by methods described herein or known to one of skill in the art(e.g., BrdU incorporation assay). In specific embodiments, a method foran inhibiting or antagonizing KIT activity in cells expressing KITcomprises contacting the cells with an effective amount of an antibodydescribed herein sufficient to inhibit cell proliferation by at leastabout 1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold,3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70fold, 80 fold, 90 fold, or 100 fold as assessed by methods describedherein or known to one of skill in the art (e.g., BrdU incorporationassay).

In certain aspects, a method provided herein for inhibiting KIT activityin a cell expressing KIT comprises contacting the cell with an effectiveamount of an antibody described herein sufficient to reduce or toinhibit survival of the cell. Cell survival assays are described in theart and can be readily carried out by one of skill in the art. Forexample, cell viability can be assessed by using trypan-blue staining orother cell death or viability markers known in the art. In a specificembodiment, the level of cellular ATP is measured to determined cellviability. In specific embodiments, cell viability is measured inthree-day and seven-day periods using an assay standard in the art, suchas the CellTiter-Glo Assay Kit (Promega) which measures levels ofintracellular ATP. A reduction in cellular ATP is indicative of acytotoxic effect. In another specific embodiment, cell viability can bemeasured in the neutral red uptake assay. In other embodiments, visualobservation for morphological changes can include enlargement,granularity, cells with ragged edges, a filmy appearance, rounding,detachment from the surface of the well, or other changes. These changesare given a designation of T (100% toxic), PVH (partially toxic—veryheavy—80%), PH (partially toxic—heavy—60%), P (partially toxic—40%), Ps(partially toxic—slight—20%), or 0 (no toxicity—0%), conforming to thedegree of cytotoxicity seen. A 50% cell inhibitory (cytotoxic)concentration (IC₅₀) is determined by regression analysis of these data.

In specific embodiments, a method provided herein for inhibiting (e.g.,partially inhibiting) KIT activity in cells expressing KIT comprisescontacting the cells with an effective amount of an antibody describedherein sufficient to reduce or to inhibit survival of the cells by atleast about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% as assessed by methodsdescribed herein or known to one of skill in the art (e.g., trypan blueexclusion assay). In specific embodiments, a method provided herein forinhibiting (e.g., partially inhibiting) KIT activity in cells expressingKIT comprises contacting the cells with an effective amount of anantibody described herein sufficient to reduce or to inhibit survival ofthe cells by at least about 25%, 35%, 45%, 55%, or 65%, as assessed bymethods described herein or known to one of skill in the art (e.g.,trypan blue exclusion assay). In specific embodiments, a method providedherein for inhibiting KIT activity in cells expressing KIT comprisescontacting the cells with an effective amount of an antibody describedherein sufficient to reduce or to inhibit survival of the cells by atleast about 1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60fold, 70 fold, 80 fold, 90 fold, or 100 fold as assessed by methodsdescribed herein or known to one of skill in the art (e.g., trypan blueassay).

In a specific embodiment, a method provided herein for inhibiting (e.g.,partially inhibiting) KIT activity in cells expressing KIT comprisescontacting the cells with an effective amount of an antibody describedherein sufficient to induce apoptosis (i.e., programmed cell death).Methods for detecting apoptosis are described in the art and can bereadily carried out by one of skill in the art. For example, flowcytometry can be used to detect activated caspase 3, anapoptosis-mediating enzyme, in cells undergoing apoptosis, or Westernblotting can be used to detect cleavage of poly(ADP-ribose) polymerase(PARP (see, e.g., Smolich et al., Blood, 2001, 97:1413-1421). Cleavageof PARP is an indicator of apoptosis. In specific embodiments, a methodprovided herein for an inhibiting or antagonizing KIT activity in cellsexpressing KIT comprises contacting the cells with an effective amountof an antibody described herein sufficient to induce or enhanceapoptosis by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% asassessed by methods described herein or known to one of skill in the art(e.g., flow cytometry to detect activated caspases 3). In specificembodiments, a method provided herein for an inhibiting or antagonizingKIT activity in cells expressing KIT comprises contacting the cells withan effective amount of an antibody described herein sufficient to induceor enhance apoptosis by at least about 25%, 35%, 45%, 55%, or 65%, asassessed by methods described herein or known to one of skill in the art(e.g., flow cytometry to detect activated caspases 3). In specificembodiments, antibodies a method provided herein for inhibiting KITactivity in cells expressing KIT comprises contacting the cells with aneffective amount of an antibody described herein sufficient to induce orenhance apoptosis by at least about 1 fold, 1.2 fold, 1.3 fold, 1.4fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 foldas assessed by methods described herein or known to one of skill in theart (e.g., flow cytometry to detect activated caspase 3).

In a specific embodiment, a method provided herein for inhibiting (e.g.,partially inhibiting) KIT activity in a cell expressing KIT comprisescontacting the cells with an effective amount of an antibody describedherein sufficient to induce differentiation. Methods for detectingdifferentiation are described in the art and can be readily carried outby one of skill in the art. For example, flow cytometry can be used todetect expression of one or more differentiation markers, or the lack ofexpression of one or more undifferentiated markers, in a cell contactedwith an antibody described herein. Similarly, Western blotting can alsobe used to detect differentiation markers. Suitable differentiationmarkers and undifferentiated markers have been described and are one ofskill in the art.

In specific embodiments, a method provided herein for inhibiting (e.g.,partially inhibiting) KIT activity in cells expressing KIT comprisescontacting the cells with an effective amount of an antibody describedherein sufficient to induce differentiation by at least about 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 98%, or 99% as assessed by methods described herein orknown to one of skill in the art (e.g., flow cytometry). In specificembodiments, a method provided herein for inhibiting (e.g., partiallyinhibiting) KIT activity in cells expressing KIT comprises contactingthe cells with an effective amount of an antibody described hereinsufficient to induce differentiation by at least about 25%, 35%, 45%,55%, or 65%, as assessed by methods described herein or known to one ofskill in the art (e.g., flow cytometry). In specific embodiments, amethod provided herein for inhibiting KIT activity in cells expressingKIT comprises contacting the cells with an effective amount of anantibody described herein sufficient to induce differentiation by atleast about 1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60fold, 70 fold, 80 fold, 90 fold, or 100 fold as assessed by methodsdescribed herein or known to one of skill in the art (e.g., flowcytometry).

Non-limiting examples of cells which can be differentiated by themethods described herein include stem cells (e.g., embryonic stem cells,hematopoietic stem cells) and progenitor cells. Exemplary hematopoieticstem cell markers include CD38, CD34, CD59, CD133, Sca-1, and ABCG2.Non-limiting examples of neural stem cell markers include Nestin,PSA-NCAM, p75 Neurotrophin R, and Vimentin. Other non-limiting examplesof stem cell markers include, Oct4, Sox2, Klf4, LIN28, Nanog, SSEA-3,SSEA-4, Notch, and Wnt.

6. EXAMPLES

The examples in this section (i.e., section 6) are offered by way ofillustration, and not by way of limitation.

6.1 Example 1: Generating Murine Anti-KIT Monoclonal Antibodies

Murine monoclonal antibodies that immunospecifically bind to the D4/D5region of human KIT were obtained from immunized mice using hybridomatechnology. From this process, antibody 37M was isolated.

Briefly, two female HTP™ mice (Abpro, Inc., Cambridge, Mass.) wereimmunized with a KIT immunogen corresponding to the D4/D5 fragment ofhuman KIT (see FIG. 2, in particular FIG. 2A depicting the recombinantantigen (containing the D4/D5 region of human KIT)). Prior toimmunization, the KIT immunogen was emulsified in Freund's CompleteAdjuvant. Booster immunizations also were emulsified in Freund'sIncomplete Adjuvant.

Antibody serum titers were determined after the 8th immunization on Day18 by ELISA, and fusion was performed on Day 21 to generate hybridomacells. Lymphocytes were obtained from the lymph nodes of one selectedimmunized mouse. Hybridoma cells were generated by fusing thelymphocytes with NS0 myeloma fusion partner cells using the HTP™protocol. The hybridoma cells were divided into eight 96-well plates forscreening.

A primary screen was performed with IgG-specific secondary antibody toidentify hybridoma clones secreting IgG antibodies. Up to 48 sampleswere selected for expansion in 24-well plates.

Thirty-two pre-subclones were screened a second time for binding to theKIT immunogen (i.e., the D4/D5 region of human KIT) by ELISA and forblocking KIT phosphorylation by cell based assays, which are describedin more detail in the sections below. Clones that reached a 25%inhibition determined by cell based phosphorylation assays were rankedand the 10 best clones were selected for subcloning.

Twenty subclones were tested for binding to the KIT immunogen by ELISA,and for blocking KIT phosphorylation by cell based assays. The best twoclones were also tested by fluorescence-activated cell sorting (FACS)for binding to full-length KIT expressed on CHO cells. From thesescreening procedures, antibody 37M was isolated.

The sequences of the light chain and heavy chain of antibody 37M wereobtained by PCR methods. The sequences of the variable light chainregion and the variable heavy chain region, as well as the sequences ofthe CDRs and framework regions (FRs) were also determined based on theKabat numbering system.

The DNA fragments encoding the VL chain region and the VH chain regionof antibody 37M were cloned into the cassette vectors containing humanconstant region of a heavy chain (IgG1) and a light chain (Kappa)resulting in the antibody 37C, which is a chimeric antibody consistingof murine variable regions and human constant regions

FIG. 3A shows the amino acid sequence of the VL domain of antibodies 37Mand 37C. FIG. 3B shows the amino acid sequence of the VH domain ofantibodies 37M and 37C. The CDR and FR amino acid sequences are labeledaccordingly. FIG. 4 shows the amino acid sequences of the VL and VHdomains of antibody 37M as well as the nucleic acid sequences encodingthem. FIG. 5A shows the amino acid sequence of the light chain and heavychain of antibody 37C. Both the light chain (SEQ ID NO: 6) and heavychain (SEQ ID NO: 7) include a signal peptide, variable regioncontaining CDRs and FRs, and a constant region. The signal peptide iscleaved during post-translational processing, and is not present in themature form of the light chain and heavy chain. FIG. 5B shows thenucleotide sequence (SEQ ID NO: 10) encoding the light chain of antibody37C, and the corresponding amino acid sequence (SEQ ID NO: 6). FIG. 5Cshows the nucleotide sequence (SEQ ID NO: 11) encoding the heavy chainof antibody 37C, and the corresponding amino acid sequence (SEQ ID NO:7).

6.2 Example 2: Antibodies 37M and 37C have High Affinity for the D4/D5Region of Human KIT

The binding activity of antibodies 37M and 37C obtained from miceimmunized with a KIT immunogen containing the D4/D5 region of the humanKIT extracellular region was characterized by solid phase ELISA.Specifically, the binding affinity of antibody 37M for antigenscontaining the D4 region (see FIG. 2B), D5 region (see FIG. 2C), or theD4/D5 region (see FIG. 2A) of human KIT was tested by solid phase ELISA.The binding affinity of antibody 37C for an antigen containing the D4/D5region (see FIG. 2A) of human KIT was also tested by solid phase ELISA.The general protocol used for the solid phase ELISA experiments aredescribed below.

Materials:

-   -   Recombinant antigen: Recombinant IG domain four and five of the        extracellular region of KIT    -   Assay plates: Nunc MaxiSorp micro-titer plates 456537    -   plate sealers Fisher 353073    -   polypropylene dilution plates    -   TBS-T: 50 mM Tris pH 7.4, 150 mM NaCl, 0.1% Tween 20    -   TBS: 50 mM Tris pH 7.4, 150 mM NaCl    -   Blocking solution: 1% bovine serum albumin (BSA) in TBS    -   Dilution buffer: 1% BSA in TBS-T    -   Detection antibody solution: Goat anti-mouse IgG HRP antibody        and Pierce goat anti-human F(ab′)₂ specific conjugated with        horseradish peroxidase (Thermo scientific 31414)    -   Detection Substrate: TMB (3,3′,5,5′-tetramethylbenzidine)        Substrate kit (Thermo scientific #34021)    -   Plate Washer: NUNC Immunowash 470176    -   ELISA plate reader: BioTek Synergy HT, Software Gen5 1.08

Coating plates with antigen: Recombinant antigens corresponding to theD4 region (see FIG. 2B), D5 region (see FIG. 2C), or D4/D5 region of theKIT extracellular domain (see FIG. 2A) were absorbed onto 96-wellmicrotiter plates. Recombinant antigen (10 μg) were diluted into 10 mLof borate buffer, and 100 μL of the antigen solution were added to eachwell of a 96 well plate. The 96 well plate was covered with a platesealer, was incubated at 4° C. overnight, and then stored at −80° C.until the day of the assay.

Preparation of antibody sample with serial dilution: Dilution buffer(100 μl was added to columns 1 and 7 of a 96 well plate, and 50 μL ofdilution buffer were added to all the remaining wells. The test antibody(100 μl was added to the first well containing 100 μL dilution buffer,mixed by pipetting, and 50 μL from the first well were removed and addedinto the second well and mixed. This dilution step was repeated acrossthe row until the dilution step was repeated for a total of sixdilutions. This dilution process was repeated for each sample.

ELISA: The plate with the absorbed antigen was removed from the −80° C.freezer and was allowed to thaw at room temperature. The borate bufferwas removed by flicking the plate into the sink and blotting the platedry. Blocking buffer (200 μl was added to each well and was allowed toincubate at room temperature for one hour. The blocking buffer wasremoved, again by flicking the plate into the sink. Then, the dilutedsolution of test antibodies and controls were added to the plate in avolume of 50 μL and incubated at room temperature for one hour. Theantibody solutions were removed, and the plate was washed four timeswith 300 μL of wash buffer with five minute incubations. After the lastwash, the plate was blotted dry. Secondary antibody solution was diluted1:8000 and was added to each well in a volume of 100 μL and allowed toincubate for one hour at room temperature. The diluted secondaryantibody solution was removed, and the plate was washed four times with300 μL of wash buffer with five minute incubations. The plate was thenblotted dry, and freshly mixed TMB substrate solution was added to eachwell in a volume of 100 μL and was allowed to incubate at roomtemperature for 30 minutes. Subsequently, 100 μL of 2N H₂SO₄ were addedto each well and immediately read on the plate reader. An irrelevantantibody served as the negative control, and an anti-KIT antibodyagainst the D4 and/or D5 domain of the extracellular region of KITserved as the positive control. OD values for each sample were obtainedat a wavelength of 450 nm.

Data analysis using Graph Pad Prism and Excel: Concentrations in ng/mLunits were converted to μM based on the molecular weight of a Fabmolecule (50 kDa) and of an IgG molecule (150 kDa). The OD values fromthe plate reader for the samples were exported into Graph Pad and thesample concentrations were transformed into log(X) and subjected tononlinear regression (curve fit), then sigmoidal dose-response (variableslope) to obtain the concentration of antibodies at 50% binding toantigen.

FIG. 6A depicts a graph plotting OD450 versus log concentration (nM) ofantibody 37M. The effective concentration at 50% binding (EC₅₀) for thebinding affinity of antibody 37M to the D4/D5 region of human KIT wascalculated to be approximately 192 pM. The EC₅₀ for the binding affinityof antibody 37M to the D4 region and to the D5 region of human KIT wascalculated to be approximately 572 pM and greater than 3 μM,respectively. The results indicate that antibody 37M has high affinityto domain D4 and the entire antigen (D4/D5 region of KIT), but not todomain D5 alone.

FIG. 6B depicts a graph plotting OD450 versus log concentration (nM) ofantibody 37C. The EC₅₀ for the binding affinity of antibody 37C to theD4/D5 region of human KIT was calculated to be approximately 196 pM. Theresults indicate that antibody 37C has high affinity for the D4/D5region of human KIT. The results presented in FIGS. 6A and 6B show thatthe binding affinities of mouse antibody 37M and chimeric antibody 37Care comparable.

To confirm that antibody 37M can bind to KIT expressed on the surface ofcells, flow cytometry assays were carried out using CHO cells that do(CHO/KIT-WT) and do not (parental CHO cells) exogenously express thefull-length, wild-type human KIT receptor. Briefly, parental CHO cellsand CHO/KIT-WT cells were washed and incubated with 0.01 nM, 0.1 nM, 1nM or 10 nM of antibody 37M, a negative control isotype IgG antibody, ora commercial anti-KIT antibody as a positive control. The samples wereprocessed for flow cytometry analysis. More specifically, cells wereremoved from the culture flasks using EDTA, and washed with PBS. Then,the cells were resuspended in media and counted. Each sample containingapproximately 200,000 to 250,000 cells was spun, the media was removed,and the cells were resuspended in FC buffer (1% BSA, 0.01% sodium azidein 1×PBS) for the blocking step. The cells were incubated in FC bufferfor 1 hour on ice. Then, primary antibody (e.g., antibody 37M, positivecontrol anti-KIT antibody, or negative control antibody) was added tothe cells. The samples were mixed and incubated on ice for 1 hour,followed by washing the cells with 0.5-1 mL FC buffer. The FC buffer wasremoved by spinning the cells at 1000 rpm for 5 minutes at 4° C.,decanting the liquid. The cell pellets were resuspended in 200 μL FCbuffer, and secondary antibody (DyLight 488 AffiniPure Goat Anti-MouseIgG Jackson Laboratories) was added to the cells at a 1:1000 to 1:2000dilution. The samples were mixed and incubated on ice for 1 hour, andthen washed as described above. The samples were run on a fluorescenceactivated cell sorter (FACS) machine (Accuri FlowCytometer). Sampleswere analyzed by following channel FLA-1 for DyLight-conjugated samples.

FIG. 7 depicts the results from the flow cytometry analysis. Inparticular, more intense fluorescent signals were detected in sampleswhere the CHO/KIT-WT cells were incubated with increasing concentrationsof antibody 37M, while no such increase in fluorescent signals weredetected in samples where CHO parental cells were incubated withincreasing concentrations of antibody 37M. These results indicate thatantibody 37M is able to bind to KIT expressed on the surface of cells.

Similar flow cytometry experiments were carried out with antibody 37Mand CHO/KIT-WT cells sorted for high KIT expression. The following cellsort protocol was performed. CHO cells were transfected with KIT DNAconstructs encoding full-length wild-type human KIT and a G418resistance cassette. For selection of the highest KIT-expressingpopulation, transfected CHO cells were stained for FACS analysis andsorted based on KIT expression. Briefly, parental CHO and transfectedCHO cells were collected with the use of 2 mM EDTA and washed one timewith FACS buffer (PBS+1% NBFCS+0.01% sodium azide). Approximately 10×10⁶cells were blocked in 1 mL FACS buffer on ice for 1 hr. Cells were thenspun down and re-suspended in 3 mL FACS buffer plus anti-KIT-PE antibody(Dako, 1:200), placed on ice for 1 hr, followed by 2×1 mL washes withFACS buffer. Finally, cells were washed once with 2 mL Sort buffer(PBS+0.1% sodium azide) and re-suspended in 1 mL Sort buffer. UsingFACSAria™ (BD Biosciences) instrumentation for analysis, PE fluorescenceon KIT-negative, parental CHO cells ranged from 10² to 10³. Therefore,to select for a population of highly KIT-positive cells, gating was setat ≥10⁴ (FIG. 10A, see gate P5). The sorted cells (gate P5 in FIG. 10A)were collected in CHO media containing G418 (1 mg/mL) and placed innormal culture conditions for future use.

FIG. 10C depicts the results from a flow cytometry experiment which wascarried out with sorted (high KIT expression) CHO/KIT-WT cells. Inparticular, more intense fluorescent signals were detected in sampleswhere the CHO/KIT-WT cells (FIG. 10C) were incubated with increasingconcentrations of antibody 37M, while no such increase in fluorescentsignals was detected in samples where CHO parental cells (untransfectedCHO cells) (FIG. 10B) were incubated with increasing concentrations ofantibody 37M.

6.3 Example 3: Antibodies 37M and 37C can Block KIT PhosphorylationInduced by SCF in Cell-Based Phospho-KIT Assays

To further characterize the effect of antibodies 37M and 37C on KITactivity, specifically, SCF-induced tyrosine phosphorylation of thecytoplasmic domain of KIT, cell-based phospho-KIT assays were carriedout as follows.

Materials:

-   -   CHO cells stably transfected with a plasmid encoding full-length        human KIT (see FIG. 1), which was cloned from a human ovary cDNA        library (OriGene, Rockville, Md.)    -   Complete cell culture media (see Table 5)    -   Starving media: Cell culture media described in Table 5 without        FBS    -   Trypsin-EDTA (Cellgro 25-050-CI)    -   PBS (GIBCO 10010-023)    -   24-well cell culture plates BD Falcon (353226)    -   ELISA plates (Nunc, #436110)    -   SCF solution: rhSCF (RD Systems 255-SC/CF); final concentration        30 ng/mL    -   Lysis buffer: 50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 1%        Triton X-100, protease inhibitor cocktail tables EDTA free        (Roche Diagnostics 04693132001), 1 mM NaVO₄    -   TBS-T: 50 mM Tris pH 7.4, 150 mM NaCl, 0.1% Tween 20    -   Blocking solution: 5% bovine serum albumin (BSA) in TBS-T    -   Dilution buffer: 1% BSA in TBS-T containing 1 mM NaVO₄    -   Detection antibody solution: anti-phospho-tyrosine antibody        conjugated with horse radish peroxidase (Millipore, 4G10);        dilution factor 1:500    -   Capture antibody: anti-CD117 antibody Ab3 from Thermo Scientific        (MS-289-PABX)    -   Cell incubator: Thermo Scientific, HeraCell 150i CO₂ incubator    -   Cell Counter: Invitrogen Countess™ C10227    -   ELISA plate reader: BioTek Synergy HT, Software Gen5 1.08

TABLE 5 Cell Culture Media Cell line CHO (parental or KIT transfected)Basic medium Gibco F12 Nutrient Mixture (Ham) 1X 11765Penicillin/Streptomycin 50 IU/mL penicillin (Cellgro 30-001-CI) 50 μg/mLstreptomycin 100X GlutaMAX ™-I 1X GlutaMAX ™ (Gibco 35050) Geneticin(Invitrogen 1 mg/mL Geneticin (for selection of 10131027) transfectedcells only)

Passaging of CHO/KIT-WT cells: Confluent cells were washed once withsterile PBS, incubated with 0.25% Trypsin-EDTA at room temperature untilcells detached from the plastic tissue culture plates. Complete culturemedium, which contains FBS, was added to the plate to end the trypticdigestion.

Counting Cells: Ten microliters of cell suspension were mixed with 10 μLof 0.4% trypan blue. Half of this mixture (10 μL) was transferred into acell counting chamber (Invitrogen), and the cells were counted. Cells(200,000 per well) were transferred into a 24-well cell culture plate,and were cultivated in complete medium (Table 5) for 24 hours undernormal cell culture conditions (i.e., humidified 95% air and 5% CO₂atmosphere at 37° C.).

Cell Treatment: After the cells were plated in the 24-well plates andcultured overnight, the medium was removed, and the cell monolayer waswashed once with starvation medium. The cells were then cultured for 24hours in starvation medium under normal cell culture conditions. Thenthe cells were treated with antibody 37M or 37C or control antibodysolutions for 2 hours under normal cell culture conditions. The finalconcentration for the antibody solution was 100 nM (5 μg/mL) or less.Subsequently, SCF solution was added to the cells pretreated withantibody 37M or 37C or control antibody at a final concentration of 30ng/mL for 10 minutes under normal cell culture conditions.

Controls:

-   -   Negative controls: starved, untreated and non-stimulated cells    -   Positive control: starved, untreated and SCF-stimulated cells    -   Drug control: starved cells, treated with 1 μM Gleevec and        stimulated with SCF    -   Antibody control: cells starved, treated with 100 nM blocking        antibody (purified mouse anti-human KIT antibody (BioLegend        A3C6E2) that binds to the SCF binding site)

Preparation of cell lysates: After stimulation, cells in the 24-wellplate were placed on ice immediately, the cells were washed once withcold PBS, and lysed with 100 μL of cold lysis buffer.

Preparation of 96-well ELISA plate with capture antibody: Captureantibody (5 μL) was diluted in 10 mL 50 mM Borate buffer, and thecapture antibody solution (100 μL or 50 ng/well) was added to each wellof the 96-well ELISA plate. The 96-well plate was incubated at roomtemperature for 5-6 hours or overnight at 4° C. The capture antibodysolution was removed prior to the blocking step. Blocking was carriedout by adding 100 μL of blocking solution to each well and allowed toincubate at room temperature for 1 hour. The blocking solution wasremoved, the wells were washed once with dilution buffer, and 50 μL ofdilution buffer were added to each well.

Phospho-KIT assay: 50 μL of the cell lysates of each sample from a wellof the 24-well plate were transferred into 1 well of the prepared96-well plate containing 50 μL dilution buffer, and the 96-well platewas incubated overnight at 4° C. Following the overnight incubation, thesupernatant was removed, and the plate was washed 3 times (5 minuteincubation each time) with TBS-T. Detection antibody dilution (100 μL)was added to each well and incubated for 1 hour at room temperature inthe dark. The plate was washed 3 times with TBS-T, washed once with TBS,and the TBS was removed. The “SuperSignal West Dura Extended DurationSubstrate” reagents (Thermo Scientific) were mixed (1:1), and 100 μL ofthe mix were added to each well.

Luminescence was detected in the ELISA plate reader using the Gen5protocol “Luminescence Glow” and the data were analyzed using MicrosoftExcel. Both antibodies 37M and 37C inhibited KIT phosphorylation inthese phospho-KIT assays.

FIG. 8 depicts a graph plotting the data from these experiments. Thegraph is a plot of arbitrary luminescence units versus log concentration(M) of either antibody 37M or antibody 37C. The 50% inhibitionconcentrations (IC₅₀) of antibody 37M and antibody 37C were calculatedto be approximately 109 pM and 167 pM, respectively. FIG. 11A depictsthe results of a separate experiment carried out with antibody 37M andCHO/KIT-WT cells. The IC₅₀ value in this experiment was calculated to beapproximately 96 pM. The results indicate that antibodies 37M and 37Care effective inhibitors of ligand (SCF)-induced tyrosinephosphorylation of the cytoplasmic domain of KIT.

Cell-based phospho-KIT assays also were carried out with a population ofCHO cells expressing wild-type KIT (CHO/KIT-WT cells) and of CHO cellsexpressing the KIT V560D mutant (CHO-V560D-KIT), wherein the cellpopulation was sorted for selection of the highest KIT expressing cells.The cell sort for high KIT expression was carried out essentially asdescribed in Section 6.2.

FIG. 11B depicts a graph plotting the data from cell-based phospho-KITassays with sorted (high KIT expression) CHO/KIT-WT cells. The graph isa plot of relative luminescence units versus log concentration (M) ofeither antibody 37M or antibody 37C. The IC₅₀ values of antibody 37M andantibody 37C were calculated to be approximately 315 pM and 334 pM,respectively, using sorted CHO/KIT-WT cells. The results indicate thatantibodies 37M and 37C are effective inhibitors of ligand (SCF)-inducedtyrosine phosphorylation of the cytoplasmic domain of KIT. Here, thesecalculated IC₅₀ values are higher than the IC₅₀ values described inFIGS. 8 and 11A for phosphorylation inhibition assays using CHO/KIT-WTcells that were not presorted for high expression of KIT. The variationin IC₅₀ values obtained in FIG. 8 and FIG. 11B can be due to differencesresulting from using sorted (high KIT expression) and unsortedCHO/KIT-WT cells. These data suggest that the blocking ability ofantibody 37M may be related to the cell surface expression level of KITprotein.

6.4 Example 4: Blocking Ligand-Induced AKT Phosphorylation

Antibody 37M was assayed for the ability to inhibit or block AKTphosphorylation, which is a downstream signaling event of KIT signaling.The assay was carried out as described in section 6.3 with the followingmodifications. First, a mouse anti-AKT antibody was immobilized on theELISA plates as a capture antibody. Second, the detection of AKTphosphorylation (phospho-AKT) was performed using a two-step method.After incubation of the cell lysates with the coated ELISA plate, abiotinylated mouse monoclonal antibody recognizing phospho-AKT (Ser473)was added to each well for 1 hour at room temperature at a dilution of1:500. Following this incubation and subsequent washes, the phospho-AKTantibody was detected with Protein Western C Streptavidin-HRP antibody(BioRad) at a dilution of 1:2500. The final detection step with TMBsubstrate solution was performed as described in Example 2 (Section6.2).

FIG. 13 depicts a graph plotting the data from phospho-AKT assays withsorted (high KIT expression) CHO/KIT-WT cells. The graph is a plot ofrelative luminescence units (RLUs) versus log concentration (M) ofantibody 37M. The IC₅₀ value of antibody 37M was calculated to beapproximately 138 pM based on data obtained with CHO/KIT-WT cells. Theresults indicate that antibody 37M is an effective inhibitor of ligand(SCF)-induced tyrosine phosphorylation of AKT downstream of KITsignaling.

6.5 Example 5: Animal Model Study of Anti-KIT Antibodies in TreatingCancer

The anti-tumor effects of anti-KIT antibodies described herein areconfirmed using mouse models, such as xenograft mouse models, of humantumors. Various mouse models for studying cancer have been described(see, e.g., Fernandez et al., J. Clin. Invest., 2007, 117(12):4044-4054). Below, mouse models, e.g., xenograft mouse models, derivedfrom a variety of patient-derived, human cell lines are described. Mousemodels for assessing toxicity are also described below.

Gastrointestional Stromal Tumor (GIST)

Mouse models of GIST have been described, for example, see, Fernández etal, J. Clin. Invest., 2007, 117(12): 4044-4054. For example, GIST cellsare harvested from subconfluent cultures by a brief exposure to 0.05%trypsin-EDTA (Invitrogen). Trypsinization is stopped with mediumcontaining 10% FBS. The cells are then washed twice in serum-free mediumand resuspended in serum-free HBSS (Invitrogen). Single-cell suspensionswith greater than 95% viability, as determined by Trypan blue exclusion,are used for the injections. To produce tumors, 1×10⁵ to 1×10⁷ GISTcells, for example 6×10⁶ GIST cells per 100 μl are injectedsubcutaneously into the unilateral flank of each SCID mouse (e.g.,female C.B-17/IcrHsd-Prkdc^(SCID) mice purchased from Harlan SpragueDawley Inc.; housed in facilities approved by and in accordance with theAmerican Association for Assessment and Accreditation of LaboratoryAnimal Care, the United States Department of Agriculture, the UnitedStates Department of Health and Human Services, and the NIH; and usedaccording to institutional guidelines). Five to ten mice per group inthe vehicle and anti-KIT antibody groups are used. Once tumors arepalpable (e.g., approximately 8-11 weeks from injection), mice arestarted on therapy with injections of normal saline (vehicle) oranti-KIT antibodies (e.g., 37M or 37C antibodies, including antibodydrug conjugates, for example, 37M or 37C antibody drug conjugates), forexample, daily, weekly, or bi-weekly intraperitoneal injections.Treatment is continued for a period of time, e.g., approximately 6weeks, with weekly 2-dimensional measurements of tumor size. Imagingmethods for detecting tumor size can also be used, e.g., MRI. All miceare sacrificed when the tumor size approach approximately 1.5 cm in thecontrol group. Tumors are collected, are fixed in formalin, and areanalyzed by H&E staining. Representative images are taken from eachtumor using a light microscope at ×40 and ×100 magnification.

A graph of tumor size or volume of each mouse plotted against time(e.g., days or weeks) after tumor injection is generated to ascertainthe effect of the anti-KIT antibodies on tumor growth in the micerelative to the vehicle negative control.

Non-limiting examples of GIST cells which may be used in these mousemodels include, GIST 430 cells and GIST882 cells (immortal GIST cellsthat possess a homozygous exon 13 missense mutation (i.e., K642E) in KIT(see, e.g., Tuveson et al., Oncogene, 2001, 20: 5054-5058)).

Leukemia

To study the effects of anti-KIT antibodies on leukemia, a xenograftmouse model using human leukemia cells (e.g., K562, HEL, or HL60 cells)is established essentially as described above, except that leukemiacells (e.g., K562, HEL, or HL60 cells) are injected into the miceinstead of GIST cells. In particular, the tumor cells are collected fromsubconfluent suspensions. To produce tumors, 1×10⁵ to 1×10⁷ tumor cellsper 100 μl are injected into each SCID mouse. The mice are thenrandomized into the following groups (n=5-10 per group): (a) normalsaline daily; and (b) anti-KIT antibodies (e.g., 37M or 37C antibodies,including antibody drug conjugates, for example, 37M or 37C antibodydrug conjugates). The mice are started on therapy (e.g., at day 0, 7, or14 or when tumors are detectable) with injections of normal saline(vehicle) or anti-KIT antibodies (e.g., daily, weekly, or bi-weeklyintraperitoneal injections). Treatment is continued for a period oftime, e.g., approximately 6 weeks, with weekly 2-dimensionalmeasurements of tumor size. Imaging methods for detecting tumor size canalso be used, e.g., MRI. Tumors are measured weekly during treatment andat necropsy.

A graph of tumor size or volume of each mouse plotted against time(e.g., days or weeks) after tumor injection is generated to ascertainthe effect of the anti-KIT antibodies on tumor growth in the micerelative to the vehicle negative control.

Mouse models of human leukemia also can be generated by injecting humanleukemia cells into nude mice or irradiated mice, via other routes, suchas intravenous route, and monitoring animal death as an indication ofprogression of leukemia in the presence or absence of treatment withanti-KIT antibodies. A survival curve is generated for each mouse toascertain the effect of anti-KIT antibodies on survival.

Lung Cancer (e.g., Small Cell Lung Cancer)

A xenograft mouse model using human lung cancer cells, e.g., human smallcell lung carcinoma cells (e.g., H526 cells, WBA cells, or NCI-H209cells) is established essentially as described above, except for a fewmodifications. For example, lung cancer cells (e.g., small cell lungcancer cells) are injected into mice instead of GIST cells. Lung cancercells, e.g., H526 tumor cells, are collected, and 1×10⁵ to 1×10⁷ lungcancer cells per 100 μl are injected into each mouse (e.g., SCID mouse).The mice are then randomized into the following groups (e.g. n=5-10 pergroup): (a) normal saline daily; and (b) anti-KIT antibodies (e.g., 37Mor 37C antibodies, including antibody drug conjugates, for example, 37Mor 37C antibody drug conjugates). The mice are started on therapy (e.g.,at day 0, 7, or 14 or when tumors are detectable) with injections (e.g.,daily, weekly, or bi-weekly intraperitoneal injections) of normal saline(vehicle) or anti-KIT antibodies. Treatment is continued for a period oftime (e.g., approximately 6 weeks or more), with weekly 2-dimensionalmeasurements of tumor size. Imaging methods for detecting tumor size canalso be used, e.g., MRI. Tumors are measured weekly during treatment andat necropsy.

A graph of tumor size or volume of each mouse plotted against time(e.g., days or weeks) after tumor injection is generated to ascertainthe effect of the anti-KIT antibodies on tumor growth in the micerelative to the vehicle negative control. A survival curve is generatedto ascertain the effect of the anti-KIT antibodies (e.g., antibody 37M,antibody comprising CDRs of antibody 37M, or a conjugate thereof) onanimal survival.

Mouse models for lung cancer (e.g., small cell lung cancer) have beendescribed (see, e.g., Garton et al., 2006, Cancer Res. 66(2):1015-24;and Wolff et al., 2004, Clin Cancer Res. 10:3528-3534), and may beadapted accordingly to study the effects of anti-KIT antibodiesdescribed herein.

Sarcoma

Xenograft models are established using cell lines derived from Ewing'sfamily of tumors, such as RD-ES, SK-ES-1 or SK-N-MC, orrhabdomyosarcomas, such as A-673. Cell lines are available from theAmerican Type Culture Collection (ATCC; Manassas, Va.). Generally,methods similar to those described above are utilized. For example,2.5-5×10⁶ cells are suspended with trypsin/EDTA or re-suspended in100-200 μL growth medium and implanted subcutaneously into the flank of6-8 week old immunodeficient mice (NuNu, SCID) (Charles RiverLaboratories, Wilmington, Mass.). Five to ten mice per group in both thevehicle and anti-KIT antibody groups are used. Once tumors are palpableor have reached 100-200 mm³, mice are started on therapy with injections(e.g., daily, weekly, or bi-weekly intraperitoneal injections) of normalsaline (vehicle) or anti-KIT antibodies (e.g., 37M or 37C antibodies,including anti-KIT antibody drug conjugates, for example, 37M or 37Cantibody drug conjugates). Treatment is continued for a period of time,e.g., approximately 6 weeks or more, and tumor size is evaluated (e.g.,twice weekly by way of 2-dimensional measurements). Imaging methods fordetecting tumor size can be used, e.g., MRI. Mice are sacrificed whenthe tumor size approach a certain size (e.g., approximately 1.5 cm) inthe control group. Tumors are collected, are fixed in formalin, and areanalyzed by H&E staining. Representative images are taken from eachtumor using a light microscope at, e.g., at ×40 and ×100 magnification.

A graph of tumor size or volume of each mouse plotted against time(e.g., days or weeks) after tumor injection is generated to ascertainthe effect of the anti-KIT antibodies on tumor growth in the micerelative to the vehicle negative control.

Mouse models for sarcoma (e.g., Ewing's sarcoma) have been described,for example, see the following list of publications, and may be adaptedaccordingly to evaluate the effects of anti-KIT antibodies (e.g.,antibody 37M or 37C):

-   -   Gonzälez et al., 2004, Clin Cancer Res.10(2):751-61;    -   Landuzzi et al., 2000, Am J Pathol. 157(6):2123-31 (6647 cells);    -   Merchant et al., 2002, JNCI 94(22):1673-1679 (TC71 cells);    -   Sturla et al., 2000, Cancer Res. 60(21):6160-70 (TC32 and RD-ES        cells);    -   Powis et al., 2006, Mol Cancer Ther. 5(3):630-636 (A-673 cells);    -   Watanabe et al., 2008, Hum Gene Ther. 19(3):300-10 (A-673        cells);    -   Rouleau et al., 2008, Clin Cancer Res. 14(22):7223-7236 (A-673        cells);    -   Karmakar et al, 2011, World J Oncol. 2(2):53-63 (RD-ES and        SK-N-MC cells);    -   Wang et al., 2009, In Vivo 23(6):903-9 (TC71 cells); and    -   Ikeda et al., 2010, Mol Cancer Ther. (3):653-60 (TC71 cells and        A4573 cells).

Humanized Mouse Model

Studies with anti-KIT antibodies, including anti-KIT antibody drugconjugates, for example, 37M or 37C antibodies, including 37M or 37Cantibody drug conjugates are carried out with mouse models generated byengraftment of immunodeficient mice with components of human immunesystem, e.g., humanized NSG mice (The Jackson Laboratory, Bar Harbor,Me.). Humanized NSG mice are NOD scid IL-2 receptor gamma chain knockoutmice (NSG) engrafted with human hematopoietic stem cells (hCD34⁺ cells)to reconstitute a human immune system.

These mice can serve as a platform for studying toxicity of anti-KITantibodies. For example, groups of mice (e.g., 1-5 mice) are injectedwith various concentrations of anti-KIT antibodies over a period of time(e.g., 4-16 weeks). The mice are assessed for toxicity indicators, e.g.,body weight, survival length.

6.6 Example 6: Competitive Binding Assays

Competitive binding assays were carried out with antibody 37M or 37C asdescribed below and using solid phase ELISA as described in Example 2(Section 6.2). In particular, serial dilutions of the reference antibody(either 37M or 37C) were combined with constant concentrations of acompeting antibody (37M in the case of 37C, and 37C in the case of 37M)and incubated in wells of 96-well plates coated with a recombinant KITpolypeptide containing the D4/D5 region depicted in FIG. 2A. Referenceantibody binding was then detected through the use of an HRP-conjugated,species-specific secondary antibody. Antibody 37M has a mouse constantregion while antibody 37C has a human constant region. Therefore, whenantibody 37M was used as a reference antibody and antibody 37C was usedas a competing antibody, the reference antibody was detected using ananti-mouse IgG secondary antibody; and when antibody 37C was used as areference antibody and antibody 37M was used as a competing antibody,the reference antibody was detected using an anti-human IgG secondaryantibody. In certain variations of these competitive binding assays, thereference antibody can be conjugated to a detection molecule, e.g.,biotin or a fluorescent molecule.

Coating plates with antigen: Recombinant antigen containing the D4/D5region of the KIT extracellular Ig-like region (see FIG. 2A) wasabsorbed to 96-well microtiter plates. Recombinant antigen, i.e., D4/D5region of human KIT (10 μg), was diluted into 10 mL of borate buffer,and 100 μL of the antigen solution were added to each well of a 96 wellplate. The 96 well plate was covered with a plate sealer and incubatedat 4° C. overnight.

Eight, 3-fold dilutions of the reference antibody samples were preparedbeginning at 20 nM (2×), and combined 1:1 with a 20 nM solution of thecompeting antibody to yield final 1× values. The plate with the absorbedantigen, was incubated with 200 μL blocking buffer at room temperaturefor one hour, prior to incubation with the reference and competitorantibodies. The control wells were incubated with buffer and referenceantibody alone at the concentrations tested. After 1 hour, the plateswere washed, and HRP-conjugated secondary antibody (anti-mouse-HRP oranti-human-HRP) was added to each well. The plates were processedsimilarly as described above for data collection on a plate reader. Dataanalysis was carried out using Graph Pad Prism and Excel as describedabove.

An increase in the EC₅₀ value for the reference antibody in the presenceof the competitor antibody relative to the reference antibody alonedemonstrated that antibodies 37M and 37C compete for binding to the KITD4/D5 antigen, as shown in FIGS. 14A-B. FIG. 14A depicts the resultsfrom competition assays where antibody 37M served as the referenceantibody and antibody 37C served as the competitor antibody (10 nM). Asshown in FIG. 14A, the EC₅₀ value for reference antibody 37M, which wascalculated to be 115 pM, increased to 2.8 nM in the presence of thecompetitor antibody 37C (10 nM). FIG. 14B depicts the results fromcompetition assays where antibody 37C served as the reference antibodyand antibody 37M served as the competitor antibody (10 nM). As shown inFIG. 14B, the EC₅₀ value for reference antibody 37C, which wascalculated to be 48 pM, increased to 1 nM in the presence of competitorantibody 37M (10 nM).

Variations of these competition assays include flow cytometry assays fordetecting binding of a reference antibody at various concentrations, inthe presence and absence of a competitor antibody, to cells expressingKIT on the cell surface.

6.7 Example 7: Inhibition of Colony Formation by KIT Expressing CHOCells in Soft Agar Assays

Antibody 37M was also tested for its ability to inhibit anchorageindependent cell growth in soft agar assays of CHO/KIT-WT cells. Softagar assay for colony formation is an anchorage independent growthassay, which is a useful assay for detecting malignant transformation ofcells. In vitro transformation is associated with certain phenotypicchanges such as loss of contact inhibition (cells can grow over oneanother) and anchorage independence (cells form colonies in soft agar).In general, nontransformed cells fail to grow when suspended in aviscous fluid or gel (e.g. agar or agarose), however when these cellsare transformed, they are able to grow in a viscous fluid or gel andbecome anchorage-independent. The process by which these phenotypicchanges occur, is assumed to be closely related to the process of invivo carcinogenesis.

The soft agar assays were carried out as follows. Base agar layer(containing agar and cell culture medium) was added to each well of a 96well plate. Cell agar layer (containing agar, cell culture medium andcell suspension) was added on top of the base agar layer. Anti-KITantibody 37M was diluted in cell culture medium and pipetted on top ofthe layers. The control samples did not contain any antibodies. Plateswere incubated at 37° C. and 5% CO₂ for 5-8 days in the presence orabsence of 30 ng/mL SCF. The ligand SCF and the antibody 37M (100 nM)were added concurrently to the agar.

When treatment was completed, the agar was solubilized and the cellswere lysed. The green fluorescent Cyquant® GR dye was mixed with thelysates. This dye exhibits fluorescence when bound to cellular nucleicacids. Fluorescence was measured at 480 nm excitation and 520 nmemission.

The results from the soft agar assays are depicted in FIG. 9. Antibody37M was able to inhibit soft agar formation induced by SCF.

6.8 Example 8: Antibody 37M Binds to Mutant KIT Containing a Duplicationof Ala502 and Tyr503

In certain cases, a somatic mutation associated with GIST in theextracellular domain of KIT (exon 9 of human KIT) has been observed,wherein the Ala and Tyr residues at positions 502 and 503, respectively,are duplicated (KIT A502-Y503 duplication mutant) (see, e.g., Marcia etal., (2000) Am. J. Pathol. 156(3):791-795; and Debiec-Rychter et al.,(2004) European Journal of Cancer. 40:689-695a). Specifically, thismutation is in domain 5 (D5) of the extracellular domain of human KIT.Binding studies were carried out to characterize the specific binding ofantibody 37M to the KIT A502-Y503 duplication mutant.

For example, a FACS-based study was performed. Briefly, sorted (high KITexpression) CHO cells expressing wild-type KIT (see, e.g., FIG. 10C) orKIT A502-Y503 duplication mutant (CHO-KIT-A502-Y503) were harvested with2 mM EDTA and blocked in PBS buffer containing 1% FBS and 0.01% SodiumAzide (to prevent receptor internalization) (FACS buffer) for one houron ice. Next, cells were incubated with antibody 37M or an IgG controlantibody for one hour on ice in FACS buffer. Cells were washed two timeswith FACS buffer, and run through the ACCURI® cytometer (Ann Arbor,Mich.) for analysis with BD Cflow® software. CHO cells stablytransfected with full-length, wild-type KIT were compared to CHO cellsstably transfected with the KIT 502-503 duplication mutant. Antibody 37Mbound to the wild-type-KIT-expressing cells evidenced by a clear shiftin the fluorescence peak following staining with the anti-KIT antibodycompared to the IgG control. In staining the CHO-KIT-A502-Y503, the 37Mantibody also generated a shift in the fluorescence peak compared to IgGcontrols. These FACS data indicate that antibody 37M binds to the KIT502-503 duplication mutant, as well as to wild-type KIT, expressed oncell surfaces. FIG. 12 depicts the results from the flow cytometryexperiment which was carried out with sorted CHO-KIT-A502-Y503. Inparticular, more intense fluorescent signals were detected in sampleswhere the sorted CHO-KIT-A502-Y503 cells were incubated with increasingconcentrations of antibody 37M.

6.9 Example 9: Antibody Internalization by NIH3T3 Cells ExpressingExogenous Human, Wild-Type KIT

Immunofluorescence staining assays were carried out to determine ifantibody 37M or 37C is internalized by cells. The immunofluorescencestaining assays were carried out essentially as described below withNIH3T3 cells engineered to express exogenous human, wild-type KIT(full-length) (“NIH3T3/KIT”). Materials and reagents for theimmunofluorescence assays include the following:

-   -   Tissue culture slides: Poly-D-Lysine coated four or eight well        chamber slide    -   Antibodies: Antibody 37M conjugated with the fluorescent dye        Alexa-488 (antibody 37M-Alexa-488) and antibody 37C conjugated        with the fluorescent dye Alexa-488 (antibody 37C-Alexa-488)    -   Fixative: 4% paraformaldehyde (PFA) (store 40% PFA microscopy        grade in fridge and dilute 1:10 with PBS just before use)    -   Mounting media: Prolong Gold antifade reagent with DAPI        (4′,6-diamidino-2-phenylindole)    -   NIH3T3 cells were engineered to express exogenous human,        wild-type KIT (full-length) (“NIH3T3/KIT”)

NIH3T3/KIT cells (15,000 to 30,000 cells per well) were seeded ontotissue culture slides. The cells were cultivated for at least 24 hoursbefore starvation in media containing no fetal bovine serum overnight.Antibody 37M-Alexa488 or antibody 37C-Alexa488 was diluted in starvationmedia containing 1% bovine serum albumin and pipetted onto the celllayer. Cell layers were washed once with PBS (room temperature) 5 to 60minutes later. Cell layers were fixed for 18 minutes with fixative atroom temperature, and were washed 3 times with PBS. The cells weremounted between slide and coverslip and were kept at room temperatureovernight. Internalization of the antibody was analyzed by confocalmicroscopy.

The immunofluorescence staining assays demonstrated that antibodies 37Mand 37C bound to the surface of NIH3T3/KIT cells, and were internalizedby NIH3T3/KIT cells.

6.10 Example 10: Antibody Internalization by CHO Cells ExpressingWild-Type KIT or Mutant KIT

Immunofluorescence staining assays were carried out to assessinternalization of antibody 37M by CHO cells expressing mutant KIT(CHO/KIT-V560D/Y823D or CHO/KIT-502.503) relative to CHO cellsexpressing wild-type KIT (“CHO/KIT-WT”). Certain mutations in KIT havebeen associated with cancer, such as a mutant KIT containing aduplication of Ala502 and Tyr503 (KIT A502-Y503 duplication mutant).This mutation is in domain 5 (D5) of the extracellular domain of humanKIT. For these experiments, CHO cells were engineered to express the KITA502-Y503 duplication mutant (“CHO/KIT-502.503”). Another mutant KITthat has been associated with cancer is mutant KIT containing mutationsV560D and Y823D. Also for these experiments, CHO cells were engineeredto express KIT containing mutations V560D and Y823D(“CHO/KIT-V560D/Y823D”).

The immunofluorescence staining assays were carried out essentially asdescribed below. Materials and reagents for the immunofluorescenceassays included the following:

-   -   24 well tissue culture plates    -   12 mm, optically clear, round glass coverslips    -   Primary antibodies: antibody 37M, β-Tubulin (9F3) rabbit        monoclonal antibody (mAb) (Cell Signaling #2128)    -   Secondary antibodies: goat anti-mouse antibody conjugated to        Oregon Green® 488 (Invitrogen #011038), goat anti-rabbit        antibody conjugated to Texas Red (Invitrogen #T2767)    -   Fixative: 4% paraformaldehyde (PFA) (store 40% PFA microscopy        grade in fridge and dilute 1:10 with PBS just before use)    -   Permeabilization solution: PBS with 0.1% Triton X-100 and 0.5%        BSA, sterile filtered    -   Blocking/dilution solution: 2% BSA in PBS, sterile filtered    -   Mounting media: ProLong® Gold antifade reagent with DAPI (P36931        Invitrogen) (4′,6-diamidino-2-phenylindole)    -   Microscope slides: FisherFinest Superfrost Microscope Slides        (Fisher #22-038-103)    -   Microscope: Nikon Eclipse Ti and NIS-Elements Software    -   CHO cells engineered to express exogenous human, wild-type KIT        (full-length) (“CHO/KIT-WT”) or mutant KIT (CHO/KIT-V560D/Y823D,        CHO/KIT-502.503)

CHO cells (75,000 cells per well) were seeded into a 24-well tissueculture plate containing one round glass coverslip per well. The cellswere cultivated for at least 6 hours before overnight starvation inmedia containing no fetal bovine serum. Following starvation, theculture media was removed from the cells and antibody 37M, diluted to33.3 nM in starvation media containing 1% bovine serum albumin, wastransferred onto the cell layer at time 0 minute, 30 minutes, 45 minutesor 55 minutes to generate a time course. Cells were incubated for theindicated times under standard culture conditions (37° C. and 5% CO₂).Cell layers were washed once with PBS (room temperature) 5 to 60 minutesafter addition of the antibody. Cell layers were fixed for 20 minuteswith 4% PFA at room temperature, and were washed 3 times with PBS. Cellmembranes were permeabilized by the addition of permeabilizationsolution for 3 minutes followed by 3 washes with PBS. Blocking solutionwas added to each well, and cells were blocked for 20 minutes at roomtemperature. The β-Tubulin (9F3) rabbit mAb was diluted 1:100 indilution solution and incubated with the cell layers for 1 hour at roomtemperature followed by 2 washes with PBS and one with blockingsolution. Both secondary antibodies were diluted together at 1:200 indilution solution before being added to the cells. Cells were incubatedin secondary antibody in the dark at room temperature for 1 hourfollowed by 3 PBS washes. The cells on the coverslips were mountedagainst the glass slides using one drop of ProLong® Gold antifadereagent with DAPI and were kept at room temperature overnight.Internalization of the antibody was analyzed by fluorescence microscopyat various time point, e.g., 5 minutes and 60 minutes of exposure toantibody 37M.

The immunofluorescence staining assays demonstrated that, in all threecell populations CHO/KIT-WT cells, CHO/KIT-V560D/Y823D cells, andCHO/KIT-502.503 cells, antibody 37M bound to the surface of these cells,and were internalized by these cells. In particular, images of cellsexposed to antibody 37M show staining of membrane-associated structuresat early time points, such as at 5 minutes after exposure to antibody37M, and show staining of internal structures (e.g., vesicles) at latertime points, such as at 60 minutes after exposure to antibody 37M. Incontrast, images of cells exposed with anti-β-Tubulin antibody, as acontrol, showed staining of elongated structures throughout thecytoplasm of the cells. These results indicate that antibody 37M hasspecific affinity for wild-type KIT as well as these mutant forms of KITassociated with cancer, and can be internalized by cells expressingthese forms of KIT. Effective internalization of antibody 37M is useful,e.g., for delivering toxins to cancer cells expressing KIT, utilizingboth wild-type and mutant forms of KIT associated with cancer.

6.11 Example 11: Antibody Internalization by H526 Cells ExpressingWild-Type KIT

Immunofluorescence staining assays were performed to assessinternalization of antibody 37M by the small cell lung cancer cell line,H526 which expresses wild-type KIT. Anti-KIT 37M antibody conjugatedwith the fluorescent dye Alexa488 (37M-Alexa488) was used in theseimmunofluorescence staining assays and were shown to be internalized byH526 cells. The immunofluorescence staining assays were carried outessentially as follows.

Material and Methods:

In each well of a four chamber slide coated with poly-D-lysine, 60,000H526 cells were grown for 24 hours and then starved in the absence offetal bovine serum for another 16 hours or overnight. Cells wereincubated under normal culture conditions (37 degrees Celsius and 5%CO2) for 10 and 30 minutes in the presence of 10, 30 and 100 nM antibody37M conjugated with Alexa488 using the Alexa Fluor® 488 Protein LabelingKit *3 labelings* from Molecular Probes (A10235 Invitrogen). Followingthe antibody incubations, cells were washed with PBS, fixed for 18minutes with 4% paraformaldehyde and mounted in ProLong® Gold antifadereagent with DAPI (P36931 Invitrogen) underneath a microscope glasscover. As a control, H526 cells were incubated for 10 or 30 minutes instarvation media containing 0.2% BSA that was used to dilute theantibody. Samples were analyzed using a confocal laser scanningmicroscope.

Results:

In contrast to the control images showing background, blurryautofluorescence, antibody 37M-488 staining was visible distinctively inclose proximity to, or at the plasma membrane of H526 cells 10 minutesafter treatment with antibody 37M-488 at all three concentrations, i.e.,10, 30 and 100 nM. After 30 minutes of incubation with antibody 37M-488,staining of vesicular structures localized in the cytoplasm could bevisualized, indicating internalization of antibody 37M-488 by H526cells. The staining of these vesicular structures was more abundant andmore distinct in cells treated with 100 nM of antibody 37M-488 than incells treated with the lower concentrations. Such staining was notobserved in the control samples treated only with the dilution bufferwithout antibody 37M-488.

6.12 Example 12: Antibody 37M as an Antibody-Drug Conjugate Blocks CellProliferation

Cell proliferation experiments were performed to assess theeffectiveness of antibody 37M as an antibody-drug conjugate (ADC) foruse as an anti-cancer therapeutic. The Mab-Zap (Advanced TargetingSystems, San Diego, Calif.) saporin-secondary antibody system was usedto generate an ADC with antibody 37M. Mab-Zap is a goat anti-mouse IgGsecondary antibody that is conjugated to saporin, aribosome-inactivating protein from the seeds of the plant Saponariaofficinalis. Upon coincubation with the 37M mouse antibody, the Mab-Zapsecondary antibody binds to the 37M antibody forming, via non-covalentinteractions, an ADC that targets KIT. Once antibody 37M isinternalized, and thus Mab-ZAP also is internalized, saporin breaks awayfrom the targeting agent and inactivates the ribosomes, which causesprotein inhibition and, ultimately, cell death.

This 37M-ADC was tested on GIST cells. Specifically, results wereobtained with the GIST cell line GIST 430 (FIGS. 15A-B). GIST 430 cellsare derived from imatinib-resistant human GIST and have a heterozygousprimary KIT exon 11 (juxtamembrane region) in-frame deletion,accompanied by a heterozygous secondary exon 13 (kinase ATP-bindingregion) missense mutation (V654A) (see, e.g., Bauer et al., Cancer Res.2006, 66:9153-9161).

The cells were plated onto 96-well plates, and then were treated withvarious doses of antibody 37M (ranging from 100 fM to 100 nM) in 2% FBSmedium, generating a dose curve, and the Mab-Zap secondary antibody (25ng/100 μL or 50 ng/100 μL). Following 96 hours in culture, CellTiterGlo®reagent (Promega Madison, Wis.) was used to determine ATP content ineach well (an indirect measurement of cell proliferation).

Specifically, dose-dependent inhibition of cell proliferation wasobserved when GIST 430 cells were treated with 37M-ADC (FIG. 15A), butnot with an ADC of an antibody that specifically binds to VEGFR-2(anti-VEGFR2 mAb) (FIG. 15B). Moreover, inhibition of cell proliferationwas not observed when a GIST cell line that does not express KITprotein, the GIST 48B cell line, was treated with 37M-ADC (FIGS. 15C-D).These results indicate that the 37M-ADC can specifically inhibitproliferation of GIST cells that express KIT.

This 37M-ADC also was tested with CHO cells engineered to expresswild-type KIT, for example, CHO/WT-KIT cells, or a mutant form of KIT,for example, CHO/KIT-502.503 cells (CHO cells expressing the KIT 502-503duplication mutant) or CHO/KIT-V560D/Y823D, as follows. The cells wereplated onto 96-well plates, and then were treated with various doses ofantibody 37M (ranging from 100 fM to 100 nM) in 2% FBS medium,generating a dose curve, and the Mab-Zap secondary antibody (25 ng/100μL or 50 ng/100 μL). Following 72 hours in culture, CellTiter-Glo®reagent (Promega Madison, Wis.) was used to determine ATP content ineach well (an indirect measurement of cell proliferation). In parallelexperiments, CHO cells were treated with an ADC of anti-VEGFR-2 mAb.

The results from these experiments are depicted in FIGS. 16A-B(CHO/WT-KIT cells), FIGS. 16C-D (CHO/KIT-502.503 cells), and FIGS. 16E-F(CHO/KIT-V560D/Y823D cells). Specifically, the results show thatinhibition of cell proliferation was observed when CHO cells expressingwild-type KIT or a mutant form of KIT (i.e., KIT-502.503 orKIT-V560D/Y823D) were treated with 37M-ADC, but not with an ADC of ananti-VEGFR2 mAb. These results indicate that 37M-ADC can specificallyinhibit proliferation of cells that express wild-type KIT or at leastcertain mutant forms of KIT that have been associated with cancer.

6.13 Example 13: Antibody 37M Binds to Denatured KIT

Experiments described herein above demonstrate that antibody 37M bindsnative, cell surface expressed KIT. An immunoblot assay was performed toexamine the ability of antibody 37M to bind denatured human KIT. Theresults of this experiment indicate that antibody 37M binds to denaturedKIT protein. Briefly, CHO-WT KIT cells were lysed, and total cell lysatewas denatured for 15 minutes at 75° C. and run on a 4-12% Bis-TrisSDS-PAGE gel in MOPS buffer. Protein was then transferred tonitrocellulose membranes. The membranes were blocked in 5% milk in TBS-Tfor 1 hour at room temperature and stained with either 1 μg/mL antibody37M or 10 μg/mL antibody 37M and rabbit monoclonal anti-β-Tubulinantibody (Cell Signaling Technology®), which was used as a loadingcontrol, overnight in TBST+1% BSA at 4° C. Following washes in TBST, themembranes were stained with anti-rabbit-horseradish peroxidase (HRP) andanti-mouse-HRP secondary antibodies for 1 hour in TBST+1% BSA. Signalswere detected with Pierce Supersignal® West Pico reagent. A bandcorresponding to the molecular weight of wild-type, full-length KIT wasdetected on the membrane immunoblotted with antibody 37M. For example,FIG. 17D is a Western blot, performed under denaturing conditions,showing that antibody 37M was able to detect KIT protein in denaturedform.

6.14 Example 14: Affinity for Glycosylated Forms of KIT

To characterize the binding specificity of antibody 37M for glycosylatedKIT and non-glycosylated KIT, solid phase ELISAs were performed.Briefly, KIT antigen containing either the entire extracellular domain(D1-D5) or domains 4 and 5 (D4/5) was de-glycosylated using PNGase F(New England Biolabs #P0705). KIT antigen was combined with 1 μL of 10×glycoprotein denaturing buffer and water to a final volume of 10 μL, anddenatured at 100° C. for 10 minutes. Next, 2 μL of 10×G7 reactionbuffer, 2 μL of 10% NP40, 5 μL water and 1 μL PNGase F were added to thereaction before incubation at 37° C. for 1 hour. PNGase F-treatedantigens were assessed for loss of glycosylation by SDS-PAGE. The shiftof the protein to a lower molecular weight in the PNGaseF-treatedsamples compared to untreated proteins indicates loss of proteinglycosylation (FIG. 17A). PNGase F-treated KIT ECD and KIT D4/D5antigens, and corresponding non-treated antigens, were used for solidphase ELISA with the 37M antibody as described in Example 2 (Section6.2). The results presented in FIGS. 17B-C demonstrate that the 37Mantibody binds with similar affinity to both glycosylated andnon-glycosylated KIT D4/D5 (FIG. 17B) or KIT ECD antigens (FIG. 17C).

To further confirm binding of the 37M antibody to both glycosylated andnon-glycosylated KIT protein, immunoprecipitation assays were performed.CHO cells transfected and selected to stably express wildtype KITprotein as described above were collected and lysed in 50 mM Tris pH 7.4with 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, protease inhibitorcocktail tablets-EDTA free, and 1 mM NaVO₄. The protein concentrationwas determined by a standard Bradford Assay, and two pools of 2 mg totalprotein were incubated with rotation overnight at 4° C. with 10 μgantibody 37M in 1.7 mL microcentrifuge tubes. Antibody 37M-bound KITprotein was immunoprecipitated from each sample using protein G magneticsepharose beads (GE Healthcare #28-9440-08) and the manufacturer'sprotocol. Following three washes of the beads with PBS, 2.5 μL of 10×denaturing buffer (New England Biolabs), 0.25 μL of 100× proteaseinhibitor mix (Roche Diagnostics) and 22.25 μL of water were added toeach tube. Samples were boiled for 10 minutes. Following denaturation, 5μL of 10× G7 reaction buffer, 5 μL of NP-40 and 9.75 μL of water wereadded to each tube. For the control (glycosylated) sample, 5 μL of waterwas then added, and for the de-glycosylated sample, 5 μL of PNGase F(NEB #P0705) was added. Samples were incubated for 1.5 hours at 37° C.The magnetic beads were then collected by centrifugation, and thesupernatants (50 μL) were transferred to new tubes and combined with 17μL 4× loading dye and 6 μL 10× sample reducing agent. For a secondelution from the magnetic beads, 25 μL 1× loading dye/reducing agent wasadded to each tube and samples were incubated at 95° C. for 10 minutes.Control samples, PNGase F-treated samples and 50 μg input (cell lysate)were run on a NuPAGE 4-12% Bis-Tris gel (Invitrogen) in 1×MOPS buffer.Protein was then transferred to a nitrocellulose membrane and a Westernblot for KIT protein and β-tubulin was performed as described above. TheWestern blot is shown in FIG. 17D. These data demonstrate that antibody37M binds both glycosylated KIT protein (input and untreated samples,higher molecular weight KIT band) and non-glycosylated KIT protein(PNGase F samples, lower molecular weight KIT band).

The embodiments described herein are intended to be merely exemplary,and those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, numerous equivalents to thespecific procedures described herein. All such equivalents areconsidered to be within the scope of the present invention and arecovered by the following claims. Furthermore, as used in thisspecification and claims, the singular forms “a,” “an” and “the” includeplural forms unless the content clearly dictates otherwise. Thus, forexample, reference to “an antibody” includes a mixture of two or moresuch antibodies, and the like. Additionally, ordinarily skilled artisanswill recognize that operational sequences must be set forth in somespecific order for the purpose of explanation and claiming, but thepresent invention contemplates various changes beyond such specificorder.

All references cited herein are incorporated herein by reference intheir entirety and for all purposes to the same extent as if eachindividual publication or patent or patent application was specificallyand individually indicated to be incorporated by reference in itsentirety for all purposes.

Other embodiments are within the following claims.

What is claimed:
 1. An antibody, which immunospecifically binds to aD4/D5 region of human KIT (SEQ ID NO: 15), comprising: (i) a variablelight (“VL”) chain region comprising a VL CDR1, VL CDR2, and VL CDR3having the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQID NO: 22, respectively; and (ii) a variable heavy (“VH”) chain regioncomprising a VH CDR1, VH CDR2, and VH CDR3 having the amino acidsequences of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25,respectively.
 2. The antibody of claim 1, which immunospecifically bindsto a D4/D5 region of human KIT (SEQ ID NO: 15), comprising: (A) a VLchain region comprising the amino acid sequence of SEQ ID NO: 2, and aVH chain region comprising the amino acid sequence of SEQ ID NO: 3; or(B) a VL chain region comprising the amino acid sequence of SEQ ID NO: 2and a VH chain region comprising the amino acid sequence of SEQ ID NO:5.
 3. The antibody of claim 1, which immunospecifically binds to a D4/D5region of human KIT (SEQ ID NO: 15), comprising: a VL chain regioncomprising a VL CDR1, VL CDR2, and VL CDR3 having the amino acidsequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22,respectively; and a VH chain region comprising the amino acid sequenceof SEQ ID NO: 3 or
 5. 4. The antibody of claim 1, whichimmunospecifically binds to a D4/D5 region of human KIT (SEQ ID NO: 15),comprising: a VL chain region comprising the amino acid sequence of SEQID NO: 2; and a VH chain region comprising a VH CDR1, VH CDR2, and VHCDR3 having the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 24,and SEQ ID NO: 25, respectively.
 5. The antibody of claim 1, whichimmunospecifically binds to a D4/D5 region of human KIT (SEQ ID NO: 15),wherein said antibody competes for binding to the D4/D5 region of humanKIT with an antibody comprising: a VL chain region comprising the aminoacid sequence of SEQ ID NO: 2, and a VH chain region comprising theamino acid sequence of SEQ ID NO: 3 or
 5. 6. An antibody, whichimmunospecifically binds to a D4/D5 region of human KIT (SEQ ID NO: 15),wherein said antibody binds to the same epitope as the epitope of anantibody comprising: a VL chain region comprising the amino acidsequence of SEQ ID NO: 2, and a VH chain region comprising the aminoacid sequence of SEQ ID NO: 3 or
 5. 7. An antibody, whichimmunospecifically binds to a D4/D5 region of human KIT (SEQ ID NO: 15),wherein said antibody competes for binding to the D4/D5 region of humanKIT with an antibody comprising: (i) a VL chain region comprising a VLCDR1, VL CDR2, and VL CDR3 having the amino acid sequences of SEQ ID NO:20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; and (ii) a VH chainregion comprising a VH CDR1, VH CDR2, and VH CDR3 having the amino acidsequences of SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25,respectively.
 8. An antibody, which immunospecifically binds to a D4/D5region of human KIT (SEQ ID NO: 15), wherein said antibody binds to thesame epitope as the epitope of an antibody comprising: (i) a VL chainregion comprising a VL CDR1, VL CDR2, and VL CDR3 having the amino acidsequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22,respectively; and (ii) a VH chain region comprising a VH CDR1, VH CDR2,and VH CDR3 having the amino acid sequences of SEQ ID NO: 23, SEQ ID NO:24, and SEQ ID NO: 25, respectively.
 9. The antibody of any one ofclaims 1 to 8, wherein said antibody further comprises a human lightchain constant region comprising the amino acid sequence of SEQ ID NO:12 and a human heavy chain constant region comprising the amino acidsequence of SEQ ID NO:
 13. 10. The antibody of any one of claims 1 to 8,wherein the antibody further comprises a human light chain constantregion and a human heavy chain constant region.
 11. The antibody ofclaim 10, wherein the human light chain constant region is a human kappalight chain constant region.
 12. The antibody of claim 10, wherein thehuman heavy chain constant region is a human gamma heavy chain constantregion.
 13. The antibody of any one of claims 1 to 12, wherein theantibody is a humanized monoclonal antibody.
 14. The antibody of any oneof claims 1 to 13, wherein the antibody is a monoclonal antibody or anisolated antibody.
 15. The antibody of any one of claims 1 to 13,wherein the antibody is a human IgG1 or IgG4 antibody.
 16. The antibodyof claim 15, wherein the antibody is an IgG1 isotype f antibody.
 17. Theantibody of any one of claims 1 to 16, wherein the antibody is aninhibitor of KIT activity.
 18. The antibody of claim 17, wherein theantibody inhibits KIT tyrosine phosphorylation by at least 25% asdetermined by a solid phase ELISA assay.
 19. The antibody of claim 18,wherein the antibody inhibits KIT tyrosine phosphorylation by 25% to 80%as determined by a solid phase ELISA assay.
 20. The antibody of claim17, wherein the antibody inhibits KIT receptor phosphorylation by atleast 50% as determined by a solid phase ELISA assay.
 21. The antibodyof claim 17, wherein the antibody does not block KIT ligand binding toKIT.
 22. The antibody of claim 17, wherein the antibody does not inhibitKIT receptor dimerization.
 23. The antibody of claim 17, wherein theantibody does not inhibit KIT receptor dimerization.
 24. The antibody ofclaim 17, wherein the antibody enhances KIT receptor internalization orKIT receptor degradation.
 25. The antibody of claim 17, wherein theantibody induces apoptosis when a cell expressing KIT is contacted withan effective amount of the antibody.
 26. The antibody of any one ofclaims 1 to 16, wherein said antibody is internalized by a cell.
 27. Aconjugate comprising the antibody of any one of claims 1 to 26, or aKIT-binding fragment thereof, linked to a therapeutic agent.
 28. Theconjugate of claim 27, wherein the therapeutic agent is a toxin.
 29. Theconjugate of claim 28, wherein the toxin is abrin, ricin A, pseudomonasexotoxin, cholera toxin, or diphtheria toxin.
 30. The conjugate of anyone of claims 27 to 29, wherein the conjugate is internalized by a cell.31. A pharmaceutical composition comprising the conjugate of any one ofclaims 27 to 30 and a pharmaceutically acceptable carrier.
 32. Anpolynucleotide comprising nucleotide sequences encoding a VH chainregion, a VL chain region, or both a VL chain region and a VH chainregion, of an antibody of any one of claims 1 to
 16. 33. Thepolynucleotide of claim 32, wherein the polynucleotide comprises anucleotide sequence encoding a VH chain region, a VL chain region, orboth a VL chain region and a VH chain region, of an antibody comprising:(i) a VL chain region comprising a VL CDR1, VL CDR2, and VL CDR3 havingthe amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO:22, respectively; and (ii) a VH chain region comprising a VH CDR1, VHCDR2, and VH CDR3 having the amino acid sequences of SEQ ID NO: 23, SEQID NO: 24, and SEQ ID NO: 25, respectively.
 34. The polynucleotide ofclaim 32, wherein the polynucleotide comprises a nucleotide sequenceencoding a VH chain region, a VL chain region, or both a VL chain regionand a VH chain region, of an antibody comprising: (i) a VL chain regioncomprising the amino acid sequence of SEQ ID NO: 2; and (ii) a VH chainregion comprising the amino acid sequence of SEQ ID NO: 3 or
 5. 35. Thepolynucleotide of claim 32, wherein the polynucleotide comprises anucleotide sequence encoding a VH chain region, a VL chain region, orboth a VL chain region and a VH chain region, of an antibody whichimmunospecifically binds to a D4/D5 region of human KIT (SEQ ID NO: 15),wherein said polynucleotide comprises the nucleotide sequence of SEQ IDNO: 8 or 9, or SEQ ID NOs: 8 and
 9. 36. The polynucleotide of claim 32,wherein the polynucleotide comprises a nucleotide sequence encoding aheavy chain, a light chain, or both a light chain and a heavy chain, ofan antibody which immunospecifically binds to a D4/D5 region of humanKIT (SEQ ID NO: 15), wherein said polynucleotide comprises thenucleotide sequence of SEQ ID NO: 10 or 11, or SEQ ID NOs: 10 and 11.37. An expression vector comprising the polynucleotide of any one ofclaims 32 to
 36. 38. The expression vector of claim 37, which is amammalian expression vector.
 39. A host cell comprising the expressionvector of claim 37 or 38 or one or more polynucleotides of any one ofclaims 32-36.
 40. A hybridoma cell producing an antibody of any one ofclaims 1 to
 16. 41. A pharmaceutical composition comprising the antibodyof any one of claims 1 to 26 and a pharmaceutically acceptable carrier.42. A kit comprising the antibody of any one of claims 1 to
 26. 43. Akit comprising the conjugate of any one of claims 27 to
 30. 44. A methodfor treating or managing a KIT-mediated disorder, comprisingadministering to a subject in need thereof a therapeutically effectiveamount of the antibody of any one of claims 1 to
 26. 45. A method fortreating or managing a KIT-mediated disorder, comprising administeringto a subject in need thereof a therapeutically effective amount of theconjugate of any one of claims 27 to
 30. 46. The method of claim 44 or45, wherein the KIT-mediated disorder is cancer, an inflammatorycondition, or fibrosis.
 47. The method of claim 46, wherein the canceris leukemia, chronic myelogenous leukemia, lung cancer, small cell lungcancer, or gastrointestinal stromal tumors.
 48. The method of claim 46,wherein the cancer is refractory to treatment by a tyrosine kinaseinhibitor.
 49. The method of claim 48, wherein the tyrosine kinaseinhibitor is imatinib mesylate or SU11248.
 50. The method of any one ofclaims 44 to 49, wherein the method further comprises administering asecond therapeutic agent.
 51. The method of claim 50, wherein the secondtherapeutic agent is a chemotherapeutic agent, tyrosine kinaseinhibitor, an antibody, or a cytokine.
 52. The method of claim 51,wherein the tyrosine kinase inhibitor is imatinib mesylate or SU11248.53. A method for diagnosing a subject with a KIT-mediated disordercomprising contacting cells or a sample obtained from the subject withthe antibody of any one of claims 1 to 16 and detecting the expressionlevel of KIT in the cells or the sample.
 54. The method of claim 53,wherein the antibody is conjugated to a detectable molecule.
 55. Themethod of claim 54, wherein the detectable molecule is an enzyme, afluorescent molecule, a luminescent molecule, or a radioactive molecule.56. A method for inhibiting KIT activity in a cell expressing KITcomprising contacting the cell with an effective amount of the antibodyof any one of claims 1 to
 16. 57. A method for inducing or enhancingapoptosis in a cell expressing KIT comprising contacting the cell withan effective amount of the antibody of any one of claims 1 to
 16. 58. Amethod for inducing cell differentiation comprising contacting a cellexpressing KIT with an effective amount of the antibody of any one ofclaims 1 to
 16. 59. The method of claim 58, wherein the cell is a stemcell.
 60. An antibody, which specifically binds to an epitope within aD4/D5 region of human KIT (SEQ ID NO: 15), comprising a light chain anda heavy chain, wherein the light chain comprises the amino acid sequenceof SEQ ID NO: 6 or the amino acid sequence of SEQ ID NO: 6 starting atposition 20, lacking the signal peptide.
 61. An antibody, whichspecifically binds to an epitope within a D4/D5 region of human KIT (SEQID NO: 15), comprising a light chain and a heavy chain, wherein theheavy chain comprises the amino acid sequence SEQ ID NO: 7 or the aminoacid sequence of SEQ ID NO: 6 starting at position 20, lacking thesignal peptide.
 62. A method of making an antibody whichimmunospecifically binds to a D4/D5 region of human KIT (SEQ ID NO: 15)comprising culturing the cell of claim 39 or
 40. 63. The method claim 62further comprising purifying the antibody obtained from said cell. 64.The method of claim 50, wherein the second therapeutic agent is ahistone deacetylase inhibitor.